Write a critique on the Alzheimer’s disease paper (between 750 to 900 words excluding the title page)<\/p>\n
I have to read the paper and critique it. Talk about the paper and the results and what the graphs mean. Also talk about what could’ve been done better.<\/p>\n<\/div>\n<\/div>\n<\/div>\n
ARTICLES\n<\/p>\n
APP binds DR6 to trigger axon pruning and
\nneuron death via distinct caspases
\nAnatoly Nikolaev\n<\/p>\n
1
\n, Todd McLaughlin\n<\/p>\n
2
\n, Dennis D. M. O\u2019Leary\n<\/p>\n
2
\n& Marc Tessier-Lavigne\n<\/p>\n
1\n<\/p>\n
Naturally occurring axonal pruning and neuronal cell death help to sculpt neuronal connections during development, but their
\nmechanistic basis remains poorly understood. Here we report that b-amyloid precursor protein (APP) and death receptor 6
\n(DR6, also known as TNFRSF21) activate a widespread caspase-dependent self-destruction program. DR6 is broadly
\nexpressed by developing neurons, and is required for normal cell body death and axonal pruning both in vivo and after
\ntrophic-factor deprivation in vitro. Unlike neuronal cell body apoptosis, which requires caspase 3, we show that axonal
\ndegeneration requires caspase 6, which is activated in a punctate pattern that parallels the pattern of axonal fragmentation.
\nDR6 is activated locally by an inactive surface ligand(s) that is released in an active form after trophic-factor deprivation, and
\nwe identify APP as a DR6 ligand. Trophic-factor deprivation triggers the shedding of surface APP in a b-secretase
\n(BACE)-dependent manner. Loss- and gain-of-function studies support a model in which a cleaved amino-terminal fragment
\nof APP (N-APP) binds DR6 and triggers degeneration. Genetic support is provided by a common neuromuscular junction
\nphenotype in mutant mice. Our results indicate that APP and DR6 are components of a neuronal self-destruction pathway,
\nand suggest that an extracellular fragment of APP, acting via DR6 and caspase 6, contributes to Alzheimer\u2019s disease.\n<\/p>\n
The initial formative phase of nervous system development, invol-
\nving the generation of neurons and extension of axons, is followed by
\na regressive phase in which inappropriate axonal branches are pruned
\nto refine connections, and many neurons are culled to match the
\nnumbers of neurons and target cells1\u20133. The loss of neurons and
\nbranches also occurs in the adult after injury, and underlies the
\npathophysiology of many neurodegenerative diseases1,4.\n<\/p>\n
Our understanding of regressive events in development remains
\nfragmentary. Degeneration can result \u2018passively\u2019 from the loss of
\nsupport from trophic factors such as nerve growth factor (NGF)1\u20133.\n<\/p>\n
There is also evidence for \u2018active\u2019 mechanisms in which extrinsic
\nsignals trigger degeneration by means of pro-apoptotic receptors,
\nincluding some members of the tumour necrosis factor (TNF) recep-
\ntor superfamily such as p75NTR (also known as NGFR), Fas and
\nTNFRSF1A (previously known as TNFR1) (Fig. 1a)5. However, the
\nfull complement of degeneration triggers remains incompletely
\nunderstood.\n<\/p>\n
Our understanding of the intracellular mechanisms of neuronal
\ndismantling is also incomplete. It is well documented that devel-
\nopmental neuronal cell body degeneration requires the apoptotic\n<\/p>\n
1Division of Research, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA. 2Molecular Neurobiology Laboratory, The Salk Institute, 10010 North Torrey Pines
\nRoad, La Jolla, California 92037, USA.\n<\/p>\n
0%\n<\/p>\n
1%\n<\/p>\n
a E10.5 E11.5 E12.5\n<\/p>\n
P P
\nPVC\n<\/p>\n
S\n<\/p>\n
M M
\nM\n<\/p>\n
V
\nC\n<\/p>\n
S\n<\/p>\n
V\n<\/p>\n
C\n<\/p>\n
S\n<\/p>\n
b\n<\/p>\n
e d 100
\n80
\n60
\n40
\n20\n<\/p>\n
NGF-deprived\n<\/p>\n
Sensory axons\n<\/p>\n
NGF-deprived
\n+ anti-DR6.1\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn
\ne
\nra\n<\/p>\n
ti
\nn\n<\/p>\n
g
\n a\n<\/p>\n
xo
\nn
\n b\n<\/p>\n
u
\nn
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n)\n<\/p>\n
+ NGF\n<\/p>\n
100
\n80
\n60
\n40
\n20\n<\/p>\n
TF-deprived\n<\/p>\n
Motor axons\n<\/p>\n
TF-deprived
\n+ anti-DR6.1\n<\/p>\n
1.5%\n<\/p>\n
+ TFs\n<\/p>\n
100
\n80
\n60
\n40
\n20\n<\/p>\n
48 h, control\n<\/p>\n
Commissural axons\n<\/p>\n
48 h, + anti-DR6.1\n<\/p>\n
24 h, control\n<\/p>\n
+
\n N\n<\/p>\n
G
\nF\n<\/p>\n
\nN\n<\/p>\n
G
\nF\n<\/p>\n
-d
\ne
\np\n<\/p>\n
ri
\nve\n<\/p>\n
d<\/p>\n
Sensory, TuJ1\n<\/p>\n
+
\n A\n<\/p>\n
n
\nti
\n-D\n<\/p>\n
R
\n6
\n.1\n<\/p>\n
\n+\n<\/p>\n
I
\ng\n<\/p>\n
G<\/p>\n
+
\n T\n<\/p>\n
F
\ns\n<\/p>\n
+
\n I
\ng\n<\/p>\n
G<\/p>\n
+
\n A\n<\/p>\n
n
\nti
\n-D\n<\/p>\n
R
\n6
\n.1\n<\/p>\n<\/p>\n
Motor, p75NTR\n<\/p>\n
T
\nF\n<\/p>\n
-d
\ne
\np\n<\/p>\n
ri
\nve\n<\/p>\n
d<\/p>\n
f\n<\/p>\n
2
\n4
\n h\n<\/p>\n<\/p>\n
GFP TUNEL\n<\/p>\n
4
\n8
\n h\n<\/p>\n
\n+\n<\/p>\n
A
\nn
\nti
\n-D\n<\/p>\n
R
\n6
\n.1\n<\/p>\n
\n+\n<\/p>\n
I
\ng\n<\/p>\n
G<\/p>\n
+
\n I
\ng\n<\/p>\n
G<\/p>\n
Commissural\n<\/p>\n
c\n<\/p>\n
?
\nNeuro-
\ntrophins FasL\n<\/p>\n
Fasp75NTRDR6\n<\/p>\n
TNF\n<\/p>\n
TNFRSF1A\n<\/p>\n
0\n<\/p>\n
0\n<\/p>\n
0\n<\/p>\n
Figure 1 | DR6 regulates degeneration of several
\nneuronal classes. a, Diagram of several TNF
\nreceptor superfamily members possessing death
\ndomains. b, DR6 mRNA is expressed by
\ndifferentiating spinal neurons (including motor
\n(M) and commissural (C)), and by sensory (S)
\nneurons in DRG at E10.5\u2013E12.5. Expression is
\nlow in neuronal progenitors (P) in the ventricular
\nzone (V). c\u2013f, Anti-DR6.1 (50 mg ml\n<\/p>\n
21
\n) reduces\n<\/p>\n
degeneration in vitro. c, Anti-DR6.1 inhibits
\ncommissural axon degeneration (visualized with
\ngreen fluorescent protein (GFP), right) and cell
\nbody death (TUNEL labelling, left; dots indicate
\nexplants) seen after 48 h in dorsal spinal cord
\ncultures. Red arrow indicates degenerating axon.
\nd, e, Effect on degeneration of sensory (d) or
\nmotor (e) axons triggered by trophic deprivation.
\nTFs, trophic factors (BDNF and NT3). Axons
\nwere visualized by immunostaining for tubulin
\n(TuJ1; sensory) or p75NTR (motor). f, The
\npercentage of degenerating axon bundles in
\nc\u2013e (mean and s.e.m., n 5 3 replicates). Scale
\nbars, 110 mm (b, c) and 50 mm (d, e).\n<\/p>\n
Vol 457 | 19 February 2009 | doi:10.1038\/nature07767\n<\/p>\n
981
\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n
effectors BAX and caspase 3 (refs 6\u20138); pruning of a particular dend-
\nrite in Drosophila is also caspase-dependent9,10. Developmental axo-
\nnal degeneration similarly has many hallmarks of apoptosis\u2014
\nincluding blebbing, fragmentation, and phagocytic clearing of debris
\nby neighbouring cells2,4. However, it has been argued that axonal
\ndegeneration is caspase-independent, because caspase 3 inhibitors
\nblock cell body but not axonal degeneration8 (reflecting higher
\nactivation of caspase 3 in cell bodies compared to axons11), and
\nbecause genetic manipulations to inhibit apoptosis did not block
\naxonal degeneration in some models12,13. These results indicated
\nthe existence of a caspase-independent program of axonal degenera-
\ntion1,2,4, but its molecular nature has remained elusive.\n<\/p>\n
While studying the expression of all TNF receptor superfamily
\nmembers14, we found that DR6\u2014one of eight members possessing a
\ncytoplasmic death domain (Fig. 1a)\u2014is widely expressed by neurons
\nas they differentiate and enter a pro-apoptotic state. DR6 is an orphan
\nreceptor15. In transfected cells, it triggers cell death in a Jun N-terminal
\nkinase-dependent manner16. In vivo, it regulates lymphocyte develop-
\nment17,18, but its involvement in neural development is unknown.\n<\/p>\n
Here we show that DR6 links passive and active degeneration
\nmechanisms. After trophic deprivation, DR6 triggers neuronal cell
\nbody and axon degeneration. Because DR6 signals via BAX and cas-
\npase 3 in cell bodies, we revisited the involvement of caspases in
\naxonal degeneration, and found that axonal degeneration indeed
\nrequires both BAX and a distinct effector, caspase 6. Our results also
\nindicated that DR6 is activated by a prodegenerative ligand(s) that is
\nsurface-tethered but released in an active form after trophic depriva-
\ntion. In searching for candidate ligands with these properties, we
\nconsidered APP, a transmembrane protein that undergoes regulated
\nshedding and is causally implicated in Alzheimer\u2019s disease19\u201322,
\nbecause we had previously found it to be highly expressed by devel-
\noping neurons and especially axons (see later). Because Alzheimer\u2019s
\ndisease is marked by neuronal and axonal degeneration, we had long
\nwondered whether APP participates in developmental degeneration.
\nWe show that an extracellular fragment of APP is indeed a ligand for
\nDR6\u2014as is a fragment of its close relative APLP2\u2014that triggers
\ndegeneration of cell bodies via caspase 3 and axons via caspase 6,
\nand we propose that this developmental mechanism is hijacked in
\nAlzheimer\u2019s disease.\n<\/p>\n
DR6 regulates neuronal death\n<\/p>\n
To explore the involvement of the TNF receptor superfamily in
\nneural development, we screened its 28 members by in situ hybrid-
\nization in midgestation mouse embryos. We came to focus on DR6
\n(Fig. 1a), because its messenger RNA is expressed at low levels in
\nproliferating progenitors in the spinal cord, but is highly expressed
\nby differentiating neurons within the spinal cord and adjacent dorsal
\nroot ganglia (DRG) (Fig. 1b).\n<\/p>\n
Because DR6-expressing neurons are becoming dependent for sur-
\nvival on trophic support at these stages, we examined whether DR6
\nregulates neuronal death after trophic-factor deprivation in vitro,
\nfocusing on three sets of spinal neurons: commissural, motor and
\nsensory (Supplementary Fig. 1a). Initially, we found that short inter-
\nfering RNA (siRNA) knockdown of DR6 protected commissural
\nneurons from degeneration (Supplementary Fig. 2). This prompted
\nus to screen monoclonal antibodies to DR6 for their ability to mimic
\nthis protection; we selected antibody 3F4 (anti-DR6.1). When
\nembryonic day (E)13.5 rat dorsal spinal cord explants are cultured
\nfor 24 h, commissural cell bodies and axons are healthy, but they
\ndegenerate if cultured for 24 h longer 23; anti-DR6.1 inhibited this
\ndegeneration (Fig. 1c, f), mimicking DR6 knockdown. Anti-DR6.1
\nalso protected sensory neurons from E12.5 mouse DRGs cultured for
\n48 h with NGF, and motor neurons from E12.5 mouse ventral spinal
\ncord explants cultured for 24 h with brain-derived neurotrophic fac-
\ntor (BDNF) and neurotrophin 3 (NTF3, also known as NT3): when
\nthese cultures were deprived of trophic factor and cultured for 24 h
\nlonger, they showed massive cell death and axonal degeneration,\n<\/p>\n
which were largely inhibited by anti-DR6.1 (Fig. 1d\u2013f and
\nSupplementary Fig. 1b). Similar protection was observed when
\nDRGs or ventral explants from a DR6 null mutant17 were deprived
\nin the absence of anti-DR6.1 (Supplementary Fig. 4b and data not
\nshown), confirming that anti-DR6.1 is function-blocking. DR6
\ninhibition (by antibody, siRNA or genetic deletion) caused a delay
\nrather than a complete block, because more degeneration was
\nobserved in each case 24\u201348 h later (Fig. 2b, Supplementary Fig. 4b
\nand data not shown). Consistent with a delay, there was a higher
\nmotor-neuron number at E14.5 in the DR6 mutant, but this returned
\nto the wild-type level by E18 (Supplementary Fig. 3), after the cell
\ndeath period. Thus, antagonizing DR6 delays the death of several
\nneuronal populations in vitro and in vivo.\n<\/p>\n
DR6 regulates axonal pruning\n<\/p>\n
DR6 protein is expressed not just by cell bodies (data not shown) but
\nalso by axons (Supplementary Fig. 4a). Protection of axons by DR6\n<\/p>\n
a\n<\/p>\n
b
\n+ NGF 48 h\n<\/p>\n
C
\no\n<\/p>\n
n
\ntr\n<\/p>\n
o
\nl I\n<\/p>\n
g
\nG\n<\/p>\n
\nA\n<\/p>\n
n
\nti
\n-D\n<\/p>\n
R
\n6
\n.1\n<\/p>\n<\/p>\n
NGF-deprived\n<\/p>\n
12 h 24 h\n<\/p>\n
1%\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn\n<\/p>\n
e
\nra\n<\/p>\n
ti
\nn\n<\/p>\n
g
\n a\n<\/p>\n
xo
\nn\n<\/p>\n
b
\nu\n<\/p>\n
n
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n)\n<\/p>\n
NGF-deprived + control IgG
\nNGF-deprived + anti-DR6.1\n<\/p>\n
+ NGF\n<\/p>\n
48 h24 h\n<\/p>\n
100\n<\/p>\n
80\n<\/p>\n
60\n<\/p>\n
40\n<\/p>\n
20\n<\/p>\n
c\n<\/p>\n
d\n<\/p>\n
e\n<\/p>\n
f\n<\/p>\n
Scratch in substratum Teflon divider\n<\/p>\n
Neurites NeuritesCell bodies\n<\/p>\n
WT\n<\/p>\n
WT\n<\/p>\n
DR6\u2013\/\u2013\n<\/p>\n
DR6\u2013\/\u2013\n<\/p>\n
0\n<\/p>\n
Figure 2 | DR6 regulates axon pruning in vitro and in vivo. a, Diagram of
\nCampenot chamber (adapted from ref. 24). b, Images show the local
\ndegeneration of sensory axons (TuJ1 immunostain) in Campenot chambers
\nafter NGF deprivation from the axonal compartment (top) was delayed by
\nanti-DR6.1 (50 mg ml\n<\/p>\n
21
\n) added at the time of deprivation (bottom). The\n<\/p>\n
graph shows the percentage of degenerating bundles at 24 and 48 h (mean
\nand s.e.m., n 5 3 replicates). c\u2013f, Compromised pruning of retinal axons in
\nDR62\/2 mice.Dorsalviewof (c, e),andvibratomesectionsthrough(d, f),the
\nsuperior colliculus of wild-type (WT; c, d) or DR62\/2 (e, f) mice at P6. In
\nwild-type mice (c, d), DiI-labelled temporal RGC axons form a dense
\ntermination zone (TZ) in anterior superior colliculus (arrowheads denote
\nthe anterior border). Few are outside the immediate termination zone area
\n(arrows). In DR6\n<\/p>\n
2\/2
\nmice (e, f), temporal RGC axons and arbors are present\n<\/p>\n
in areas far from the termination zone (inset, magnified in e, right) and well
\nposterior to it (arrows). L, lateral; M, medial; P, posterior. Scale bars, 100 mm
\n(b), 400 mm (c, e, left), 170 mm (e, right) and 250 mm (d, f).\n<\/p>\n
ARTICLES NATURE | Vol 457 | 19 February 2009\n<\/p>\n
982
\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n<\/div>\n
inhibition might therefore reflect a direct role for DR6 in axons. To
\nexplore this, we used compartmented (\u2018Campenot\u2019) chambers24\n<\/p>\n
(Fig. 2a). Sensory neurons are placed in a central chamber containing
\nNGF; their axons grow under a partition into NGF-containing side-
\nchambers. Fluid exchange between the chambers is limited, so NGF
\ndeprivation in a side-chamber elicits local axon degeneration while
\nsparing cell bodies24. Locally deprived axons degenerate in a stereo-
\ntypical manner with initial signs by 6 h and extensive degeneration by
\n12\u201324 h, but when anti-DR6.1 was added to the deprived side-
\nchamber, degeneration was blocked at 24 h and still largely impaired
\nat 48 h (Fig. 2b). A similar delay was observed when axons of neurons
\nfrom DR6 knockout mice were locally deprived, but in the absence of
\nanti-DR6.1 (Supplementary Fig. 4b, c). Thus, DR6 function is required
\nin axons for degeneration.\n<\/p>\n
To determine whether DR6 functions in axonal pruning in vivo, we
\nstudied the well-characterized retino-collicular projection, which
\ndevelops from an initially exuberant projection of retinal ganglion
\ncell (RGC) axons to a focused termination zone in the superior
\ncolliculus. All temporal RGC axons initially extend into posterior
\nsuperior colliculus, well past their future termination zone in anterior
\nsuperior colliculus (Supplementary Fig. 5a). This diffuse projection is
\nthen refined by selective degeneration of inappropriate axon seg-
\nments2, such that by postnatal day (P)6 in wild-type mice few axon
\nsegments persist in areas well beyond the termination zone, as
\nrevealed by focal injection of the lipophilic dye DiI into temporal
\nretina (Fig. 2c, d and Supplementary Fig. 5a, b). In contrast, in P6
\nDR6 mutant mice, many more RGC axons and arbors are present in
\nareas far from the termination zone (Fig. 2e, f and Supplementary Fig.
\n5c, d): we found an 83% increase in axon-positive domains more
\nthan 400 mm from the termination zone (Supplementary Fig. 5f) in
\nDR6\n<\/p>\n
2\/2
\n(n 5 7) compared to wild-type mice (n 5 7; P , 0.05,\n<\/p>\n
Student\u2019s t-test). The defect at P6 represents a delay in pruning,
\nnot a complete block, as assessed by examining labelled axons at
\nP4, P5, P6 and P9: at each age, the mutant has more extraneous axons
\nthan the wild-type, and fewer are observed in both wild-type and
\nmutant at each age compared to earlier time points, but by P9 the
\nmutant and wild-type projections are indistinguishable (data not
\nshown). Thus, blocking DR6 function delays pruning of sensory
\naxons in vitro and retinocollicular axons in vivo.\n<\/p>\n
Caspase 6 regulates axonal degeneration\n<\/p>\n
Because DR6 regulates both cell body apoptosis and axonal degen-
\neration, we revisited whether an apoptotic pathway is also involved in
\naxons. In support, we found that BAX, an effector in the intrinsic
\napoptotic pathway, is required in axons, because local sensory axon
\ndegeneration in Campenot chambers was blocked by the genetic
\ndeletion of Bax (Fig. 3a) or by local addition of a BAX inhibitor
\n(for example, Supplementary Fig. 10b). Consistent with evidence
\nthat caspase 3 mediates cell body but not axon degeneration8,11, we
\nfound that procaspase 3 is highly enriched in cell bodies, and that
\nzDEVD-fmk, an inhibitor of effector caspases 3 and 7, blocked cell
\nbody but not axon degeneration (Fig. 3b, c and Supplementary Fig.
\n6a\u2013c). There is, however, a third effector caspase, caspase 6. We found
\nthat procaspase 6 is expressed in both cell bodies and axons, and that
\nthe caspase 6 inhibitor zVEID-fmk blocked degeneration of sensory,
\nmotor and commissural axons (Fig. 3b, c and Supplementary Fig.
\n6a\u2013c), suggesting that caspase 6 regulates axonal degeneration. We
\nverified these results using RNA interference in sensory and commis-
\nsural neurons: Casp3 knockdown protected cell bodies significantly
\nbut had only a minor protective effect on axons, whereas Casp6
\nknockdown protected axons significantly with only minor effect on
\ncell bodies (Fig. 3d, e). Thus, distinct caspases mediate cell body and
\naxon degeneration.\n<\/p>\n
To visualize caspase activation, we first used the fluorescent repor-
\nters FAM-DEVD-fmk (for caspase 3 and 7) and FAM-VEID-fmk (for
\ncaspase 6), which bind covalently to activated target caspases. In
\nNGF-deprived sensory neurons, the caspase 3\/7 reporter labelled cell\n<\/p>\n
bodies but not axons, consistent with a previous study11; in contrast,
\ncaspase 6 reporter labelling was observed in both cell bodies and
\naxons, and axonal labelling occurred in regularly spaced \u2018puncta\u2019,
\ngiving a beads-on-a-string appearance (Supplementary Fig. 6f). To
\ncontrol for reporter specificity, we used a selective antibody to
\ncleaved caspase 6 and observed a similar punctate pattern in axons
\n(Fig. 3f, g), whereas antibodies to cleaved caspase 3 only label cell
\nbodies11. Caspase 6 activation was confirmed biochemically
\n(Supplementary Fig. 6e). Interestingly, caspase 6 activation appeared
\nat sites of microtubule fragmentation (assessed by the loss of tubulin
\nimmunoreactivity) (Fig. 3f, g), suggesting that caspase 6 activation
\ndrives microtubule destabilization. Punctate caspase 6 activation was
\nmarkedly reduced by anti-DR6.1 (Fig. 3f) and abolished in Bax\n<\/p>\n
2\/2\n<\/p>\n
neurons (not shown), suggesting that caspase 6 acts downstream of
\nBAX in the pathway triggered by DR6. However, the possibility of
\nfeedback loops in apoptotic pathways makes this interpretation tent-
\native.\n<\/p>\n
Regulated shedding of a DR6 ligand(s)\n<\/p>\n
As DR6 is a receptor-like protein, we addressed whether it is activated
\nby a ligand(s). If so, the DR6 ectodomain might be capable of binding
\nthe ligand(s) and blocking its action (Fig. 4a). Consistent with this,
\nthe DR6 ectodomain fused to human Fc (DR6\u2013Fc) mimicked anti-
\nDR6.1 in delaying degeneration (Fig. 4a\u2013c and Supplementary Figs 7a
\nand 13). To search for DR6 binding sites on axons and in conditioned
\nmedium, we used the DR6 ectodomain fused to alkaline phosphatase
\n(DR6\u2013AP). Purple alkaline phosphatase reaction product was
\nobserved on sensory and motor axons cultured with trophic factors
\nwhen they were pre-incubated with DR6\u2013AP but not with alkaline
\nphosphatase alone, but binding was markedly reduced after trophic
\ndeprivation (Supplementary Fig. 7b, c). To control for the loss of
\naxonal membrane, we blocked degeneration using a BAX inhibitor
\n(data not shown) or using neurons from Bax\n<\/p>\n
2\/2
\nmice (Fig. 4d) and\n<\/p>\n
observed an even greater reduction in DR6\u2013AP binding (residual
\nbinding seen without BAX inhibition might reflect nonspecific bind-
\ning to degenerating axons). To determine whether DR6-binding sites
\nwere shed, we collected medium conditioned by sensory axons (in
\nCampenot chambers) or motor neurons (in explant culture) (a BAX
\ninhibitor was added to prevent nonspecific release resulting from
\ndegeneration). Proteins were separated on non-reducing gels, blotted
\nto nitrocellulose, and probed with DR6\u2013AP. Little signal was seen in
\nmedium conditioned by either neuronal type in the presence of
\ntrophic factor. However, 48 h after trophic deprivation, DR6\u2013AP
\nbound a prominent band around ,35 kDa and a minor band around
\n,100 kDa in both cultures (Fig. 4e). Together, these results support a
\n\u2018ligand activation\u2019 model in which a prodegenerative DR6 ligand(s) is
\npresent on the neuronal surface and inactive, but is shed into med-
\nium in an active form after trophic deprivation (Fig. 4f), allowing it to
\nbind and activate DR6.\n<\/p>\n
N-APP is a regulated DR6 ligand\n<\/p>\n
Several properties of APP made it a candidate for a DR6 ligand: (1) it
\nis highly expressed by developing spinal and sensory neurons and
\ntheir axons (Fig. 4g), (2) its ectodomain can be shed in a regulated
\nfashion19,20, and (3) it is tied to degeneration through its links to
\nAlzheimer\u2019s disease19\u201322. In an initial experiment, we found that
\nDR6\u2013AP bound APP expressed in COS-1 cells (Supplementary Fig.
\n8a). This prompted us to test whether the bands detected by DR6\u2013AP
\nin conditioned medium (Fig. 4e) represent APP ectodomain frag-
\nments. APP is cleaved by a- or b-secretases (including, in neurons,
\nBACE1; ref. 25) at distinct sites in its juxtamembrane region (Fig. 4h)
\nto release ,100-kDa ectodomain fragments termed sAPPa or sAPPb,
\nrespectively19,20. We probed conditioned medium with a polyclonal
\nantibody to the APP N terminus (anti-N-APP(poly), which also
\nbinds the APP relative APLP2; see later) and an antibody selective
\nfor the carboxy-terminal epitope of sAPPb exposed by BACE cleav-
\nage (anti-sAPPb) (Fig. 4h). Notably, anti-N-APP(poly) detected\n<\/p>\n
NATURE | Vol 457 | 19 February 2009 ARTICLES\n<\/p>\n
983
\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n<\/div>\n
similar bands to those detected by DR6\u2013AP: a major band at ,35 kDa
\nand a minor band at ,100 kDa, both highly enriched after trophic
\ndeprivation (Fig. 4i); anti-sAPPb detected a minor ,100-kDa band
\nand a major ,55-kDa band (Fig. 4i), also both enriched after trophic
\ndeprivation. These results indicate that trophic deprivation triggers
\nBACE cleavage of APP to yield the ,100-kDa sAPPb (detected by
\nboth antibodies), which undergoes a further cleavage(s) to yield a
\n,55-kDa C-terminal fragment (detected by anti-sAPPb) and a ,35-
\nkDa N-terminal fragment (detected by anti-N-APP(poly)) that we
\nterm N-APP. The site of additional cleavage(s) is unknown, but on
\nthe basis of fragment sizes it is expected to be around the junction
\nbetween the APP \u2018acidic\u2019 and \u2018E2\u2019 domains (amino acid 286); indeed,
\nrecombinant APP(1\u2013286) ran at ,35 kDa and was detected with
\nanti-N-APP(poly) (Fig. 4j), similar to N-APP.\n<\/p>\n
Supporting cleavage of APP by BACE, we found that APP express-
\nion on the surface of cultured sensory and motor axons, as assessed
\nwith anti-N-APP(poly) and with antibody 4G8 to the APP juxta-
\nmembrane region (Fig. 4h), is high in the presence of trophic factor\n<\/p>\n
but lost after trophic deprivation; the surface loss was blocked by
\nthree structurally divergent BACE inhibitors\u2014OM99-2, BACE
\ninhibitor IV, and the highly selective AZ29 (ref. 26) but not the
\na-secretase inhibitor TAPI (Fig. 4k, Supplementary Figs 9a\u2013c and
\n10a, and data not shown). Interestingly, 4G8 partially inhibited sur-
\nface loss (Supplementary Fig. 9d), presumably through the steric
\nhindrance of BACE. Loss of surface APP occurred progressively
\nand in \u2018patches\u2019, with little lost at 3 h, more at 6\u201312 h, and most lost
\nby 24 h (Fig. 4k, Supplementary Fig. 10b and data not shown). Total
\nAPP visualized after permeabilization did not change detectably
\n(Supplementary Fig. 10b). Surface loss was not affected by BAX or
\ncaspase 6 inhibitors, or in neurons from Bax\n<\/p>\n
2\/2
\nmice (Fig. 4k and\n<\/p>\n
Supplementary Figs 9c and 10c).
\nThe marked similarly of bands detected by anti-N-APP(poly) and\n<\/p>\n
DR6\u2013AP suggested that DR6 binds N-APP. Indeed, depletion of
\nconditioned medium with anti-N-APP(poly) eliminated DR6\u2013AP
\nbinding sites (Fig. 4i), and purified DR6\u2013Fc bound to purified
\nrecombinant APP(1\u2013286) in pull-down (Fig. 4j) and enzyme-linked\n<\/p>\n
a\n<\/p>\n
Cleaved caspase 6 TuJ1\/Cleaved caspase 6\n<\/p>\n
+
\n N\n<\/p>\n
G
\nF\n<\/p>\n
\nN\n<\/p>\n
G
\nF\n<\/p>\n
-d
\ne
\np\n<\/p>\n
ri
\nve\n<\/p>\n
d
\n +\n<\/p>\n
c
\ntr\n<\/p>\n
l I
\ng\n<\/p>\n
G<\/p>\n
N
\nG\n<\/p>\n
F
\n-d\n<\/p>\n
e
\np\n<\/p>\n
ri
\nve\n<\/p>\n
d
\n +\n<\/p>\n
a
\nn
\nti
\n-D\n<\/p>\n
R
\n6
\n.1\n<\/p>\n<\/p>\n
NGF-deprived, 24 h\n<\/p>\n
+ Caspase 6 inhibitor + Caspase 3 inhibitor\n<\/p>\n
T
\nu
\nJ1\n<\/p>\n<\/p>\n
d\n<\/p>\n
Procaspase 6 TuJ1 Procaspase 3 TuJ1\n<\/p>\n
T
\nu
\nJ1\n<\/p>\n
\/T
\nU\n<\/p>\n
N
\nE\n<\/p>\n
L\n<\/p>\n
+ NGF\n<\/p>\n
+ Casp6 siRNA1 + Casp3 siRNA1\n<\/p>\n
NGF-deprived NGF-deprived\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn
\ne
\nra\n<\/p>\n
ti
\nn
\ng\n<\/p>\n
a
\nxo\n<\/p>\n
n
\n b\n<\/p>\n
u
\nn
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n)\n<\/p>\n
+ NGF\n<\/p>\n
+ anti-NGF\n<\/p>\n
+ CASP3_si1\n<\/p>\n
+ CASP3_si2\n<\/p>\n
+ CASP3_si3\n<\/p>\n
+ CASP6_si1\n<\/p>\n
+ CASP6_si2\n<\/p>\n
+ CASP6_si3\n<\/p>\n
Casp3
\nsiRNAs\n<\/p>\n
Casp6
\nsiRNAs\n<\/p>\n
Casp3
\nsiRNAs\n<\/p>\n
Casp6
\nsiRNAs\n<\/p>\n
+ NGF
\n+ Anti-NGF
\n+ Casp3 siRNA1
\n+ Casp3 siRNA2
\n+ Casp3 siRNA3
\n+ Casp6 siRNA1
\n+ Casp6 siRNA2
\n+ Casp6 siRNA3\n<\/p>\n
S
\nu
\nrv\n<\/p>\n
iv
\nin\n<\/p>\n
g<\/p>\n
D
\nR\n<\/p>\n
G
\n n\n<\/p>\n
e
\nu
\nro\n<\/p>\n
n
\ns\n<\/p>\n
(%
\n)\n<\/p>\n
NGF-deprived, 48 h\n<\/p>\n
B
\nax\n<\/p>\n
+
\n\/+\n<\/p>\n
B
\nax\n<\/p>\n
\u2013\/
\n\u2013\n<\/p>\n
+ NGF b\n<\/p>\n
c\n<\/p>\n
f\n<\/p>\n
ge\n<\/p>\n
NGF-deprived\n<\/p>\n
NGF-deprived,
\n+ caspase 3i\n<\/p>\n
+ NGF\n<\/p>\n
NGF-deprived,
\n+ caspase 6i\n<\/p>\n
1%\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn\n<\/p>\n
e
\nra\n<\/p>\n
ti
\nn\n<\/p>\n
g
\n a\n<\/p>\n
xo
\nn\n<\/p>\n
b
\nu\n<\/p>\n
n
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n)\n<\/p>\n
100\n<\/p>\n
80\n<\/p>\n
60\n<\/p>\n
40\n<\/p>\n
20\n<\/p>\n
0\n<\/p>\n
NGF-deprived, 12 h, confocal\n<\/p>\n
T
\nu
\nJ1\n<\/p>\n
\/c
\nle\n<\/p>\n
a
\nve\n<\/p>\n
d
\n c\n<\/p>\n
a
\nsp\n<\/p>\n
a
\nse\n<\/p>\n
6<\/p>\n
0
\n10
\n20
\n30
\n40
\n50
\n60
\n70
\n80
\n90\n<\/p>\n
100\n<\/p>\n
0
\n10
\n20
\n30
\n40
\n50
\n60
\n70
\n80
\n90\n<\/p>\n
100\n<\/p>\n
Figure 3 | BAX and caspase 6 regulate axonal degeneration. a, Local
\nsensory axon degeneration (TuJ1 immunostain) 48 h after NGF deprivation
\nin Campenot chambers was blocked in neurons from Bax\n<\/p>\n
2\/2
\nmice.\n<\/p>\n
b, Dissociated sensory neurons double-labelled for procaspase 3 and TuJ1
\n(left), or procaspase 6 and TuJ1 (right). Caspase 3 is detected in cell bodies
\n(arrowheads), whereas caspase 6 is seen in both cell bodies and axons.
\nc, Local degeneration of sensory axons in Campenot chambers deprived of
\nNGF for 24 h is inhibited by a caspase 6 inhibitor (zVEID-FMK; caspase 6i),
\nbut not by a caspase 3\/7 inhibitor (zDEVD-FMK; caspase 3i). Quantification
\nis shown to the right (mean and s.e.m., n 5 3 replicates). d, In dissociated
\nsensory neuron cultures deprived of NGF for 24 h, siRNA knockdown of
\nCasp3 primarily rescues cell body death (TUNEL label), whereas Casp6
\nknockdown primarily rescues axonal degeneration. e, Quantification of data\n<\/p>\n
from d. Shown are the percentage of degenerating axon bundles (that is, still
\nvisible bundles that show breakdown) and the percentage of surviving
\nneurons (that is, TUNEL\n<\/p>\n
2
\n, TuJ1\n<\/p>\n
1
\n) (mean 6 s.e.m., n 5 3 replicates). Note\n<\/p>\n
that surviving axons may have TUNEL
\n1
\ncell bodies. The extent of inhibition\n<\/p>\n
by individual siRNAs correlates with the degree of target knockdown
\n(Supplementary Fig. 6d). f, Detection of caspase 6 activation in sensory
\naxons with a cleaved caspase-6-specific antibody (left; TuJ1 double-label on
\nright). Punctate activation of caspase 6 after NGF deprivation (16 h, middle
\npanel) was reduced by anti-DR6.1 (bottom panel). g, Confocal section of a
\nfield from f shows that active caspase 6 puncta correspond to sites of tubulin
\nloss (fraction non-overlapping: 82 6 3.5%; mean 6 s.e.m., n 5 8 fields).
\nScale bars, 75 mm (a, d), 100 mm (b), 50 mm (c, f) and 25 mm (g).\n<\/p>\n
ARTICLES NATURE | Vol 457 | 19 February 2009\n<\/p>\n
984
\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n<\/div>\n
immunosorbent assay (ELISA) (Supplementary Fig. 8c) assays. The
\ninteraction detected by ELISA is of high affinity (effector concentra-
\ntion for half-maximum response (EC50) 5 ,4.6 nM). The inter-
\naction of DR6\u2013AP with full-length APP expressed in COS cells was
\nalso of high affinity (half maximal saturation 5 ,1.3 nM)
\n(Supplementary Fig. 8a, b). This binding was blocked by anti-N-
\nAPP(poly) (data not shown) and anti-DR6.1 (Supplementary Fig.
\n8a), consistent with APP being a functional DR6 ligand.\n<\/p>\n
Antibodies 4G8 and anti-sAPPb used earlier are highly specific for
\nAPP. However, like other antibodies to the N terminus of APP27,\n<\/p>\n
anti-APP(poly) also binds the close APP relative APLP2 (data not
\nshown). We found that a recombinant N-terminal fragment of
\nAPLP2 also binds DR6 (Supplementary Fig. 11a). Thus, APLP2 might
\ncontribute with APP to the bands detected on western by DR6\u2013AP.
\nIndeed, an antibody selective for the APLP2 N terminus detected a
\nshed fragment in conditioned medium after trophic deprivation
\n(Supplementary Fig. 11b). The relative contribution of APP and
\nAPLP2 fragments to DR6\u2013AP binding sites remains to be determined.\n<\/p>\n
To evaluate receptor specificity, we examined by pull-down the
\nbinding of APP(1\u2013286) to ectodomains of the seven other\n<\/p>\n
98\n<\/p>\n
62\n<\/p>\n
38\n<\/p>\n
28\n<\/p>\n
16
\n14\n<\/p>\n
98\n<\/p>\n
62\n<\/p>\n
38\n<\/p>\n
28\n<\/p>\n
16
\n14\n<\/p>\n
+ \u2013 + \u2013 TFs
\nSA\n<\/p>\n
98\n<\/p>\n
62\n<\/p>\n
38\n<\/p>\n
28\n<\/p>\n
16\n<\/p>\n
DR6\u2013AP
\nVSC\n<\/p>\n
e f\n<\/p>\n
c d b\n<\/p>\n
T
\nu
\nJ1\n<\/p>\n
NGF-deprived\n<\/p>\n
Control Fc DR6\u2013Fc
\nControl Fc\n<\/p>\n
100\n<\/p>\n
80\n<\/p>\n
60\n<\/p>\n
40\n<\/p>\n
20\n<\/p>\n
DR6\u2013Fc\n<\/p>\n
Sensory axons\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn
\ne
\nra\n<\/p>\n
ti
\nn
\ng\n<\/p>\n
a
\nxo\n<\/p>\n
n
\nb\n<\/p>\n
u
\nn
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n)\n<\/p>\n
+ NGF NGF-deprived\n<\/p>\n
B
\nax\n<\/p>\n
-\/
\n–\n<\/p>\n
S
\ne
\nn
\nso\n<\/p>\n
ry
\n a\n<\/p>\n
xo
\nn
\ns\n<\/p>\n
DR6\u2013AP
\na\n<\/p>\n
A
\nP\n<\/p>\n
P\n<\/p>\n
E11.5 E12.5
\nDREZV\n<\/p>\n
C\n<\/p>\n
S\n<\/p>\n
VLF\n<\/p>\n
S\n<\/p>\n
VLF\n<\/p>\n
V DREZ\n<\/p>\n
SN\n<\/p>\n
g\n<\/p>\n
k\n<\/p>\n
S
\nu
\nrf\n<\/p>\n
a
\nc
\ne
\n A\n<\/p>\n
P
\nP\n<\/p>\n
Control + BACE inhibitor + TAPI\n<\/p>\n
NGF-deprived + BAX inhibitor (12 h) + NGF\n<\/p>\n
h\n<\/p>\n
22C11\n<\/p>\n
Anti-N-APP(poly)\n<\/p>\n
Anti-sAPP\u03b2\n<\/p>\n
4G8
\nAnti-A\u03b2
\n(33-42)\n<\/p>\n
SA CM\n<\/p>\n
NGF\n<\/p>\n
DR6\u2013AP\n<\/p>\n
98\n<\/p>\n
62\n<\/p>\n
38\n<\/p>\n
28\n<\/p>\n
16
\n14\n<\/p>\n
Anti-APP depletion
\nMock depletion\n<\/p>\n
i\n<\/p>\n
IP: Fc (protein A\/G)\n<\/p>\n
APP(1\u2013286)\u2013His\n<\/p>\n
Inputs: N-APP\n<\/p>\n
APP(1\u2013286) (~35 kDa)\n<\/p>\n
DR6\u2013Fc\n<\/p>\n
Heparin\n<\/p>\n
Anti-APP\n<\/p>\n
APP(1\u2013286) (~35 kDa)
\nAnti-APP\n<\/p>\n
DR6\u2013Fc\n<\/p>\n
Fc control\n<\/p>\n
Anti-Fc\n<\/p>\n
j\n<\/p>\n
98\n<\/p>\n
62\n<\/p>\n
38\n<\/p>\n
28\n<\/p>\n
16
\n14\n<\/p>\n
N-APP\n<\/p>\n
NGF + \u2013 + \u2013\n<\/p>\n
sAPP\u03b2\n<\/p>\n
98\n<\/p>\n
62\n<\/p>\n
38\n<\/p>\n
28\n<\/p>\n
16
\n14\n<\/p>\n
NGF\n<\/p>\n
Ligand?\n<\/p>\n
DR6\u2013Fc\n<\/p>\n
DR6\n<\/p>\n
+ TFs \u2013 TFs\n<\/p>\n
DR6\n<\/p>\n
Released
\nligand,
\nactive\n<\/p>\n
Surface
\nligand,
\ninactive DR6\n<\/p>\n
?
\n?\n<\/p>\n
\u2013 \u2013 \u2013+
\n\u2013 \u2013 +\u2013
\n+ \u2013 \u2013\u2013\n<\/p>\n
\u2013 \u2013 +\u2013
\n\u2013 + \u2013\u2013
\n\u2013 \u2013 ++
\n+ + ++\n<\/p>\n
CuBD\n<\/p>\n
KPI OX2\n<\/p>\n
TM\n<\/p>\n
A\u03b2\n<\/p>\n
CytoE2\/carbohydrate binding\n<\/p>\n
\u03b2-secretase \u03b3-secretase\n<\/p>\n
Acidic\n<\/p>\n
APP(1\u2013286)
\n~35 kDa\n<\/p>\n
Growth
\nfactor-like\n<\/p>\n
N C\n<\/p>\n
0\n<\/p>\n
Figure 4 | The N terminus of APP is a regulated DR6 ligand. a, Diagram of
\nthe hypothesis: if DR6 is ligand-activated, then DR6\u2013Fc might sequester
\nligand and inhibit degeneration. b, DR6\u2013Fc inhibits local degeneration of
\nsensory axons in Campenot chambers 24 h after NGF deprivation.
\nc, Quantification ofresultsin b (meanands.e.m.,n 5 3replicates).d,e, DR6-
\nbinding sites are lost from axons and released into medium after trophic
\ndeprivation. d, DR6\u2013AP binding (purple) to Bax\n<\/p>\n
2\/2
\nsensory axons (left) is\n<\/p>\n
lost 24 h after NGF deprivation (right). e, Medium conditioned by sensory
\naxons (SA) (in Campenot chambers) or ventral spinal cord explants (VSC),
\nmaintained with or deprived of trophic factors (TFs) for 24 h (sensory: NGF;
\nmotor: BDNF and NT3; BAX inhibitor present), was resolved under non-
\nreducing conditions and probed with DR6\u2013AP. The arrow indicates a major
\nband at ,35 kDa. f, Results in a\u2013e support a ligand activation model in
\nwhich an inactive DR6 surface ligand is shed in an active form after tropic
\ndeprivation. g, APP immunostaining on sections of mouse embryos at
\nindicated ages, showing neuronal and axonal expression. DREZ, dorsal root
\nentry zone; S, sensory ganglia; SN, spinal nerve; V, ventricular zone; VLF,\n<\/p>\n
ventro-lateral funiculus. h, Domain structure of APP (short form, APP695),
\nindicating b- and c-secretase cleavage sites and antibody binding sites. KPI
\nand OX2 denote alternatively spliced domains of the longer form. Adapted
\nfrom ref. 20. Ab, amyloid-b peptide; CuBD, copper binding domain; cyto,
\ncytosolic domain; TM, transmembrane domain. i, DR6 binding sites in
\nsensory axon conditioned medium (SA CM) include APP ectodomain
\nfragments. Left, anti-N-APP(poly) detects bands at ,35 kDa (major; filled
\narrow) and ,100 kDa (minor; open arrow), enriched after trophic
\ndeprivation. Middle, anti-sAPPb detects bands at ,55 kDa (major; filled
\narrow) and ,100 kDa (minor; open arrow). Right, immunodepletion using
\nanti-N-APP(poly) depletes DR6\u2013AP binding sites. Arrow indicates N-APP
\nband at ,35 kDa. j, Direct interaction between purified APP(1\u2013286) and
\nDR6\u2013Fc revealed by pull-down. The effect of heparin (10 mg ml\n<\/p>\n
21
\n) was also\n<\/p>\n
examined. IP, immunoprecipitates. k, Loss of surface APP in patches from
\nsensory axons 12 h after NGF deprivation is blocked by the BACE inhibitor
\nOM99-2 (10 mM) but not by the a-secretase inhibitor TAPI (20 mM). Scale
\nbars, 75 mm (b, d, k) and 500 mm (g).\n<\/p>\n
NATURE | Vol 457 | 19 February 2009 ARTICLES\n<\/p>\n
985
\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n<\/div>\n
death-domain-containing members of the TNF receptor superfam-
\nily, and two orphan members. Only binding to p75NTR was observed
\n(Supplementary Fig. 8d), suggesting that p75NTR might serve as an
\nalternative route for APP effects in some settings; however, the affin-
\nity was considerably lower (EC50 5 ,300 nM by ELISA;
\nSupplementary Fig. 8e). Consistent with DR6 being the chief APP
\nreceptor in our systems, a fusion of APP(1\u2013286) and alkaline phos-
\nphatase bound to sensory axons in culture, but the binding was
\nsignificantly reduced by anti-DR6.1 or by using DR6 knockout neu-
\nrons (Supplementary Fig. 12a, b); residual binding may represent
\nbackground or binding to another receptor(s), possibly p75NTR.\n<\/p>\n
Necessity and sufficiency of N-APP\n<\/p>\n
To test whether the N terminus of APP contributes to degeneration,
\nwe performed loss-of-function studies. Degeneration of sensory and
\ncommissural axons in response to trophic deprivation was inhibited
\nby anti-N-APP(poly) (Fig. 5a, d), which also inhibited the death of
\nsensory neuron cell bodies (Supplementary Fig. 13a, b), without
\naffecting the loss of surface APP after trophic deprivation
\n(Supplementary Fig. 13c). Antibody 22C11 (ref. 28) to the APP N
\nterminus (Fig. 4h) also inhibited sensory axon degeneration (data not
\nshown). Because both antibodies also bind APLP2 (ref. 27), we per-
\nformed a more selective blockade using RNA interference.
\nKnockdown of App in sensory neurons significantly impaired both
\naxon degeneration and cell body death after trophic withdrawal
\n(Fig. 5b). These results support the involvement of an N-terminal
\nfragment of APP in degeneration. In further support, BACE inhibi-
\ntors impaired degeneration of sensory axons and cell bodies (Fig. 5c
\nand Supplementary Figs 13a, b and 14) and of commissural axons
\n(Fig. 5d) after trophic deprivation. The selective BACE inhibitor
\nAZ29 blocked degeneration at concentrations consistent with its cel-
\nlular half-maximal inhibitory concentration (IC50) of 470 nM\n<\/p>\n
26\n<\/p>\n
(Supplementary Fig. 14a).\n<\/p>\n
Importantly, axonal degeneration block by BACE inhibitors could
\nbe reversed by adding purified APP(1\u2013286) to sensory (Fig. 5c) and
\ncommissural (Fig. 5d) neurons, showing that the N terminus of APP
\nis sufficient to trigger degeneration. This effect was largely blocked by
\nanti-DR6.1 (Supplementary Fig. 14b), consistent with DR6 being the
\nmost important functional receptor in these cells. Block of sensory
\ncell body degeneration by BACE inhibitors could similarly be
\nreversed by the addition of APP(1\u2013286), albeit at higher concentra-
\ntions (Supplementary Fig. 13a, b). Together, these results support the
\nmodel that shed N-APP activates DR6 to trigger degeneration.
\nDegeneration of sensory axons caused by APP(1\u2013286) in the presence
\nof BACE inhibitor was blocked if NGF was present (50 ng ml\n<\/p>\n
21
\n;\n<\/p>\n
Fig. 5c), indicating that trophic factors also inhibit signalling down-
\nstream of DR6.\n<\/p>\n
Role of amyloid-b in physiological degeneration\n<\/p>\n
BACE cleavage of APP is followed by c-secretase cleavage, yielding
\namyloid-b peptides19\u201322 (Figs 4h and 6c). Because amyloid-b peptides
\ncan be neurotoxic21,22, we examined whether they contribute to
\ndegeneration. The synthetic amyloid-b peptide Ab(1\u201342) triggered
\ndegeneration in our assays, and an antibody directed at amino acids
\n33\u201342 of amyloid-b (anti-Ab(33\u201342); Fig. 4h) blocked this effect
\n(Supplementary Fig. 9a, e), but did not block degeneration after
\ntrophic deprivation (Fig. 5e). Conversely, degeneration induced by
\nsynthetic Ab(1\u201342) was not blocked by the genetic deletion of DR6
\n(data not shown), indicating that amyloid-b operates by a mode of
\naction distinct from the physiological degeneration mechanism
\nstudied here.\n<\/p>\n
Antibody 4G8 used earlier, which binds amyloid-b residues 17\u201324
\n(Fig. 4h), also blocked the degenerative effect of Ab(1\u201342)
\n(Supplementary Fig. 9e), but unlike anti-Ab(33\u201342) it partially
\ninhibited degeneration after trophic deprivation (Fig. 5e).
\nHowever, as mentioned, 4G8 also partially inhibits the loss of surface\n<\/p>\n
a\n<\/p>\n
c\n<\/p>\n
d\n<\/p>\n
b\n<\/p>\n
+ BACE inhibitor\n<\/p>\n
+ APP(1\u2013286)\n<\/p>\n
Control\n<\/p>\n
+ NGF\n<\/p>\n
e\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn\n<\/p>\n
e
\nra\n<\/p>\n
ti
\nn\n<\/p>\n
g
\n a\n<\/p>\n
xo
\nn\n<\/p>\n
b
\nu\n<\/p>\n
n
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n)\n<\/p>\n
Control IgG\n<\/p>\n
+ APP(1\u2013286) + BACEi
\n+ BACEi\n<\/p>\n
+ APP(1\u2013286) + BACEi + NGF\n<\/p>\n
100\n<\/p>\n
80\n<\/p>\n
60\n<\/p>\n
40\n<\/p>\n
20\n<\/p>\n
Sensory axons\n<\/p>\n
100\n<\/p>\n
80\n<\/p>\n
60\n<\/p>\n
40\n<\/p>\n
20\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn
\ne
\nra\n<\/p>\n
ti
\nn
\ng\n<\/p>\n
a
\nxo\n<\/p>\n
n
\nb\n<\/p>\n
u
\nn
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n) Control IgG\n<\/p>\n
Anti-N-APP(poly)
\nBACEi
\nBACEi + APP(1\u2013286)\n<\/p>\n
100\n<\/p>\n
80\n<\/p>\n
60\n<\/p>\n
40\n<\/p>\n
20\n<\/p>\n
Control IgG\n<\/p>\n
+ Anti-N-APP(poly)\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn\n<\/p>\n
e
\nra\n<\/p>\n
ti
\nn\n<\/p>\n
g
\n a\n<\/p>\n
xo
\nn\n<\/p>\n
b
\nu\n<\/p>\n
n
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n) <\/p>\n
S
\nu
\nrv\n<\/p>\n
iv
\nin\n<\/p>\n
g
\n D\n<\/p>\n
R
\nG\n<\/p>\n
\nn
\ne
\nu\n<\/p>\n
ro
\nn\n<\/p>\n
s
\n(%\n<\/p>\n
)\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn
\ne
\nra\n<\/p>\n
ti
\nn\n<\/p>\n
g
\n a\n<\/p>\n
xo
\nn\n<\/p>\n
b
\nu\n<\/p>\n
n
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n)\n<\/p>\n
100\n<\/p>\n
80\n<\/p>\n
60\n<\/p>\n
40\n<\/p>\n
20\n<\/p>\n
+ NGF
\nNGF-deprived
\n+ App siRNA1\n<\/p>\n
+ App siRNA2
\n+ App siRNA3\n<\/p>\n
100\n<\/p>\n
80\n<\/p>\n
60\n<\/p>\n
40\n<\/p>\n
20\n<\/p>\n
100\n<\/p>\n
80\n<\/p>\n
60\n<\/p>\n
40\n<\/p>\n
20\n<\/p>\n
D
\ne
\ng\n<\/p>\n
e
\nn
\ne
\nra\n<\/p>\n
ti
\nn\n<\/p>\n
g
\n a\n<\/p>\n
xo
\nn\n<\/p>\n
b
\nu
\nn
\nd\n<\/p>\n
le
\ns\n<\/p>\n
(%
\n)\n<\/p>\n
+ NGF
\n+ Anti-NGF
\n+ 4G8
\n+ Anti-A\u03b2(33\u201342)\n<\/p>\n
Sensory axons\n<\/p>\n
Control + Anti-N-APP(poly)\n<\/p>\n
NGF-deprived, 24 h\n<\/p>\n
S
\ne
\nn\n<\/p>\n
so
\nry\n<\/p>\n
,
\nT\n<\/p>\n
u
\nJ1\n<\/p>\n<\/p>\n
+ NGF\n<\/p>\n
+ Control IgG + Anti-A\u03b2(33\u201342)+ 4G8\n<\/p>\n
NGF-deprived, 24 h\n<\/p>\n
T
\nu
\nJ1\n<\/p>\n<\/p>\n
+ BACE inhibitor + Anti-N-APP(poly) Control IgG
\n+APP(1\u2013286)\n<\/p>\n
C
\no\n<\/p>\n
m
\nm\n<\/p>\n
is
\nsu\n<\/p>\n
ra
\nl,\n<\/p>\n
4
\n8
\n h\n<\/p>\n
\nS\n<\/p>\n
e
\nn
\nso\n<\/p>\n
ry
\n a\n<\/p>\n
xo
\nn
\ns,\n<\/p>\n
T
\nu\n<\/p>\n
J1<\/p>\n
+ NGF NGF-deprived\n<\/p>\n
+ App siRNA1 + App siRNA2\n<\/p>\n
NGF-deprived\n<\/p>\n
S
\ne
\nn
\nso\n<\/p>\n
ry
\n,
\nT\n<\/p>\n
u
\nJ1\n<\/p>\n
\/T
\nU\n<\/p>\n
N
\nE\n<\/p>\n
L<\/p>\n
0\n<\/p>\n
0\n<\/p>\n
0\n<\/p>\n
0 0\n<\/p>\n
0\n<\/p>\n
Figure 5 | The APP N terminus regulates
\ndegeneration. a, Local degeneration of sensory
\naxons in Campenot chambers (NGF deprivation,
\n24 h) was blocked by anti-N-APP(poly)
\n(20 mg ml\n<\/p>\n
21
\n). Quantification is shown to the right\n<\/p>\n
for all panels (a\u2013e). b, In dissociated sensory
\nneurons, siRNA knockdown of App
\n(Supplementary Fig. 13d) significantly reduces
\naxon degeneration 24 h after trophic deprivation,
\nand partially reduces cell body death. c, Local
\ndegeneration of sensory axons in Campenot
\nchambers (NGF deprivation, 24 h) was inhibited
\nby the local addition of BACE inhibitor (BACEi)
\nOM99-2 (10 mM). Purified APP(1\u2013286) added
\nlocally restored axonal degeneration, an effect
\ninhibited by 50 ng ml\n<\/p>\n
21
\nNGF (right).\n<\/p>\n
d, Degeneration of commissural neurons and
\naxons at 48 h was inhibited by anti-N-APP(poly)
\n(20 mg ml\n<\/p>\n
21
\n) or BACE inhibitor OM99-2\n<\/p>\n
(10 mM), but restored by APP(1\u2013286). e, Effect of
\namyloid-b antibodies on sensory axon
\ndegeneration (NGF deprivation, 24 h). 4G8
\npartially inhibited, whereas anti-Ab(33\u201342) did
\nnot. Scale bars 50 mm (a, c, e), 40 mm (b) and
\n200 mm (d). All data are mean and s.e.m. for n 5 3
\nreplicates.\n<\/p>\n
ARTICLES NATURE | Vol 457 | 19 February 2009\n<\/p>\n
986
\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n<\/div>\n
APP. In contrast, the APP epitope bound by anti-Ab(33\u201342) is buried
\nin the cell membrane, so anti-Ab(33\u201342) does not bind intact APP
\nnor inhibit its surface loss (Supplementary Fig. 9a, b, d). Because
\nanti-Ab(33\u201342) does not protect, we attribute the partial protective
\neffect of 4G8 to its ability to inhibit APP shedding, not its ability to
\nblock amyloid-b toxicity. Because 4G8 does not bind APLP2, its
\nability to protect also supports the sufficiency of APP in mediating
\ndegeneration.\n<\/p>\n
Evidence for an APP\u2013DR6 interaction in vivo\n<\/p>\n
To seek evidence for an APP\u2013DR6 interaction in vivo, we examined
\nwhether the DR6 knockout exhibits any similar phenotype to those
\nreported in the App knockout\u2014or, given the potential for redund-
\nancy, in compound mutants of App with Aplp2. One phenotype
\nobserved at the neuromuscular junction in the App\n<\/p>\n
2\/2
\nAplp2\n<\/p>\n
2\/2\n<\/p>\n
double knockout is suggestive of a potential pruning defect. In
\nwild-type animals, motor axons normally terminate at synaptic sites
\n(Fig. 6a). In App\n<\/p>\n
2\/2
\nAplp2\n<\/p>\n
2\/2
\ndouble knockouts, however, there is a\n<\/p>\n
highly penetrant presence of nerve terminals past endplates29.
\nNotably, a similar phenotype was observed in the DR6 mutant: rather
\nthan terminating at endplates, many terminals were present beyond,
\ngiving characteristic finger-like protrusions (Fig. 6a, b). It is not
\nknown whether this phenotype reflects a failure to retract or excessive
\nsprouting. Nevertheless, the similarity of phenotypes supports the
\nview that APP signals via DR6 in regulating axonal behaviour in vivo.
\nIn this system, APP and APLP2 appear redundant because the axonal
\nphenotype is seen only in App\n<\/p>\n
2\/2
\nAplp2\n<\/p>\n
2\/2
\ndouble mutants, not\n<\/p>\n
single mutants29. Whether they are non-redundant in other systems
\nremains to be determined.\n<\/p>\n
Discussion\n<\/p>\n
Our results reveal a mechanism, the \u2018APP\u2013death-receptor\u2019 mech-
\nanism (Fig. 6c), in which trophic deprivation leads to the cleavage
\nof surface APP by b-secretase, followed by further cleavage of the
\nreleased fragment by an as yet unidentified mechanism (probably
\nnear the junction of APP acidic and E2 domains). This then yields\n<\/p>\n
an N-terminal ,35-kDa fragment (N-APP) which binds DR6, trig-
\ngering caspase activation and degeneration of both neuronal cell
\nbodies (via caspase 3) and axons (via caspase 6). Whether the second
\ncleavage is required for degeneration remains to be determined.
\nDegeneration induced by added APP(1\u2013286) was blocked when suf-
\nficient trophic factor was present, indicating that trophic factor not
\nonly prevents initiation of the APP cleavage cascade, but also blocks
\nsignalling downstream of DR6, providing a fail-safe mechanism to
\nprotect if DR6 is inappropriately activated in an otherwise healthy
\nneuron.\n<\/p>\n
DR6: an accelerator of self-destruction\n<\/p>\n
In all settings examined, antagonizing DR6 resulted in a delay, rather
\nthan a complete block, in neuronal death and axonal pruning. DR6 is
\ntherefore best thought of as an accelerator of degeneration\u2014neurons
\nand axons activate it for swift self-destruction when they become
\natrophic, but without it they have other, slower, ways of achieving
\nthat end, perhaps involving other pro-apoptotic receptors5 or
\nintrinsic mechanisms. This function contrasts that of the DR6 rela-
\ntive p75NTR (which can mediate degeneration when overexpressed11\n<\/p>\n
or when activated by a neurotrophin in neurons lacking the cognate
\nTRK (also known as NTRK) receptor5). p75NTR is more restricted to
\nspecific neuronal classes than DR6, and its genetic deletion provided
\nonly modest protection of sensory axons in the first 36 h after trophic
\ndeprivation (Supplementary Fig. 15), as reported previously for sym-
\npathetic axons30. In sympathetic neurons, p75NTR is thought to
\nmediate competition for NGF: cells with high NGF\/TRKA signalling
\nupregulate expression of BDNF, which acts via p75NTR to trigger
\ndegeneration of neighbouring neurons with less robust NGF\/TRKA
\nsignalling30,31. This mechanism shares with ours the expression of a
\nprodegenerative ligand(s) by the neurons themselves. However, the
\nDR6 ligand APP is activated by trophic-factor deprivation, whereas
\np75NTR ligand expression is increased by trophic-factor stimu-
\nlation31. Thus, p75NTR ligands are released by \u2018strong\u2019 neurons to
\nkill \u2018weak\u2019 neurons (a paracrine prodegenerative effect)31, whereas
\nAPP gets activated within weak neurons to accelerate self-destruction\n<\/p>\n
APP\u2013death-receptor mechanism c\n<\/p>\n
DR6+\/\u2013 DR6\u2013\/\u2013
\nN\n<\/p>\n
F
\n+\n<\/p>\n
S
\nY\n<\/p>\n
P
\n\/B\n<\/p>\n
T
\nX\n<\/p>\n
DR6+\/\u2013, high mag DR6\u2013\/\u2013, high mag\n<\/p>\n
B
\nT\n<\/p>\n
X\n<\/p>\n
a\n<\/p>\n
N
\ne
\nrv\n<\/p>\n
e
\n e\n<\/p>\n
n
\nd\n<\/p>\n
t
\ne
\nrm\n<\/p>\n
in
\na
\nl s\n<\/p>\n
p
\nro\n<\/p>\n
u
\nts\n<\/p>\n
>
\n 5\n<\/p>\n
0
\n \u00b5\n<\/p>\n
m
\n (
\n%\n<\/p>\n
)\n<\/p>\n
DR6\u2013\/\u2013
\nDR6+\/\u2013\n<\/p>\n
P0 NMJs\n<\/p>\n
9\n<\/p>\n
7\n<\/p>\n
5\n<\/p>\n
3\n<\/p>\n
1\n<\/p>\n
b\n<\/p>\n
DR6 APP\n<\/p>\n
ICD\n<\/p>\n
AICD\n<\/p>\n
Caspase\n<\/p>\n
Degeneration\n<\/p>\n
TF
\ndeprivation\n<\/p>\n
A
\n\u03b2\n<\/p>\n
A
\n\u03b2\n<\/p>\n
A
\n\u03b2\n<\/p>\n
\u03b3-secretase\n<\/p>\n
\u03b2-secretase\n<\/p>\n
?\n<\/p>\n
Figure 6 | APP and DR6 signalling: in vivo
\nevidence, and model. a, In control (DR61\/2) P0
\ndiaphragm muscle, few axons (green,
\nneurofilament (NF) and synaptophysin (SYP)
\nstain) overshoot endplates (red, fluorescent
\na-bungarotoxin (BTX) stain), and those that do
\nare short, but in DR6 mutants more overshoot
\nand many are long (arrowheads). Scale bar, 60
\nmm (left four panels) and 15 mm (right four
\npanels). b, The number of axons overshooting
\nby .50 mm (mean and s.e.m., n 5 3 wild-type, 4
\nmutants); this underestimates the effect, because
\novershooting axons are longer in mutants. NMJ,
\nneuromuscular junction. c, The APP\u2013death-
\nreceptor mechanism is shown. Trophic factor
\n(TF)-deprivation triggers the cleavage of surface
\nAPP by b-secretase, releasing sAPPb, which is
\nfurther cleaved by an unknown mechanism (\u2018?\u2019)
\nto release N-APP. This then binds DR6 to trigger
\ndegeneration through caspase 6 in axons and
\ncaspase 3 in cell bodies. Also illustrated is
\ncleavage by c-secretase to release amyloid-b (Ab)
\nand the APP intracellular domain (AICD).\n<\/p>\n
NATURE | Vol 457 | 19 February 2009 ARTICLES\n<\/p>\n
987
\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n<\/div>\n
triggered by trophic deprivation or perhaps other insults (an auto-
\ncrine prodegenerative effect).\n<\/p>\n
Caspase 6 mediates axonal degeneration\n<\/p>\n
The intracellular mechanisms of axonal degeneration and their rela-
\ntion to apoptosis have been unclear. Our results indicate that devel-
\nopmental axonal degeneration does involve an apoptotic pathway,
\nbut with a non-classical effector, caspase 6. Epistatis analysis supports
\na linear activation model from DR6 to BAX to caspase 6, but does not
\nexclude the possibility that active caspase 6 might feedback, for
\nexample, to accelerate the process; in this context, it is intriguing that
\nthe APP cytoplasmic domain is a caspase 6 substrate32. Activation of
\ncaspase 6 by trophic deprivation occurs in a punctate pattern in
\naxons, leading to a beads-on-a-string appearance, and sites of punct-
\nate caspase 6 activation correspond to sites of microtubule frag-
\nmentation. Caspase 6 might trigger microtubule destabilization by
\ncleaving microtubule associated proteins such as TAU (also known as
\nMAPT), a documented target of caspase 6 (refs 33, 34); in a recent
\nproteomic analysis, almost half the identified caspase 6 targets were
\ncytoskeleton-associated35.\n<\/p>\n
Ligands, receptors for self-destruction\n<\/p>\n
Although p75NTR also binds APP(1\u2013286), DR6 binds with a much
\nhigher affinity, and blocking DR6 function largely blocks both
\nAPP(1\u2013286) binding to sensory axons and degeneration triggered
\nby APP(1\u2013286). Thus, DR6 seems to be the major functional APP
\nreceptor in these neurons, although p75NTR might contribute in
\nother contexts. Conversely, APP may not be the only DR6 ligand:
\nAPLP2, which is coexpressed with APP in many neurons27, may also
\ncontribute to degeneration, because an N-terminal fragment is shed
\nin response to trophic deprivation, can bind DR6, and can trigger
\ndegeneration when added exogenously (Supplementary Fig. 16).
\nFuture studies will define the relative contributions of APP and
\nAPLP2 in different neuronal populations.\n<\/p>\n
The finding of similar neuromuscular junction phenotypes in DR6
\nand App\n<\/p>\n
2\/2
\nAplp2\n<\/p>\n
2\/2
\nmutants supports a ligand\u2013receptor inter-\n<\/p>\n
action, and indicates that APP and APLP2 both contribute in this
\nsystem. The aberrant axonal extensions seen could reflect an impair-
\nment of pruning, or, alternatively, a failure of axons to stop; of note,
\nthe APP ectodomain has been implicated in neurite growth inhibi-
\ntion36. Previous studies have not reported changes in neuronal cell
\ndeath in vivo in App mutants, either singly or in combination with
\nAplp1 and\/or Aplp2 mutations37,38. However, such studies did not
\nexamine spinal cord or sensory ganglia, nor involve time-course
\nanalysis to evaluate possible delays in degeneration. In vitro analysis
\nof cortical neurons from mutants has given divergent results about
\ntheir basal survival rates and susceptibility to glutamate excitotoxi-
\ncity37\u201339, but their response to trophic deprivation has not been
\nreported.\n<\/p>\n
In recent findings paralleling ours, trophic deprivation was found
\nto trigger BACE cleavage of APP in PC12 cell-derived neurons and
\nprimary hippocampal neurons, and degeneration was reduced by
\nApp knockdown (in PC12 cells) and BACE inhibition40,41. The pro-
\ndegenerative function of APP was, however, attributed to amyloid-b,
\nbecause antibodies to amyloid-b inhibited degeneration40,41. We too
\nobserved protection by antibody 4G8, but attribute this to the ability
\nof 4G8 to bind full-length APP and inhibit cleavage, because a dif-
\nferent anti-amyloid-b antibody that does not bind native APP inhib-
\nited neither shedding nor physiological degeneration, but blocked
\nthe toxic action of added amyloid-b. Conversely, the toxic effect of
\namyloid-b was not blocked by DR6 inhibition. It was also found that
\na c-secretase inhibitor, which reduced amyloid-b production after
\nBACE cleavage, inhibited degeneration40,41. We too found that
\nc-secretase inhibitors can partially inhibit degeneration of commis-
\nsural and sensory axons (Supplementary Fig. 17), but c-secretase has
\nmany substrates, and it is possible that the efficient activation of DR6
\nsignalling requires a distinct c-secretase-dependent process. Thus,\n<\/p>\n
our results argue against the involvement of amyloid-b in initiating
\nDR6-dependent degeneration in the neurons studied here, but this
\ndoes not exclude its possible involvement in other neurons, or at later
\ntimes in these neurons to augment the effects of APP\u2013DR6 signalling.\n<\/p>\n
APP\u2013DR6 signalling and neurodegeneration\n<\/p>\n
APP is expressed in adult brain and upregulated in damaged axons42.
\nDR6 is also highly expressed in adult brain (Supplementary Fig. 18).
\nGiven our findings, it is reasonable to assume that the APP\u2013death-
\nreceptor mechanism might contribute to adult plasticity, or to neu-
\nrodegeneration after injury or in disease. Interestingly, DR6 is upre-
\ngulated in injured neurons43, raising the question as to whether
\noverexpressed DR6 in neurons can trigger ligand-independent
\ndegeneration, as reported for p75NTR11.\n<\/p>\n
Given the genetic evidence linking APP and its cleavage to
\nAlzheimer\u2019s disease, we propose that signalling of APP via DR6
\n(and possibly p75NTR) may in particular contribute to the initiation
\nor progression of Alzheimer\u2019s disease, either alone or in combination
\nwith other proposed APP-dependent mechanisms, such as amyloid-
\nb toxicity21,22 or effects of the APP intracellular domain44. Of note,
\nprevious studies showed immunoreactivity for the APP N terminus
\nassociated with Alzheimer\u2019s plaques45,46, DR6 maps to chromosome
\n6p12.2-21.1, near a putative Alzheimer\u2019s disease susceptibility
\nlocus47, and sites of DR6 mRNA expression in adult brain correlate
\nin an intriguing way with known sites of dysfunction in Alzheimer\u2019s:
\nvery high in hippocampus, high in cortex, but low in striatum
\n(Supplementary Fig. 18), and high in forebrain cholinergic neu-
\nrons48, for instance. In addition, activated caspase 6, a downstream
\nDR6 effector, is associated with plaques and tangles in Alzheimer\u2019s
\ndisease, and with mild cognitive impairment34,49, consistent with the
\npossible activation of caspase 6 in neuritic processes by the APP\u2013
\ndeath-receptor mechanism (caspase 6 is also implicated in
\nHuntington\u2019s disease50). Although these results are compatible with
\nthe involvement of APP\u2013DR6 signalling in Alzheimer\u2019s, it is less clear
\nhow the mechanism fits with genetic evidence implicating altered
\nc-secretase processing in this disease19\u201322. However, the fact that
\nc-secretase inhibitors antagonize DR6-dependent degeneration hints
\nat a possible relationship.\n<\/p>\n
Thus, further study is required to determine the full implications
\nof the APP\u2013death-receptor mechanism in development, adult physi-
\nology and disease. Nonetheless, our results already tie APP to a new
\nmechanism for neuronal self-destruction in development, and sug-
\ngest that the APP ectodomain, acting via DR6 and caspase 6, con-
\ntributes to the pathophysiology of Alzheimer\u2019s disease.\n<\/p>\n
METHODS SUMMARY\n<\/p>\n
Antibodies to the following targets were used: procaspase 3 (1:200, Upstate),\n<\/p>\n
active capsase 3 (1:200, R&D), procaspase 6 (1:200, Stratagene), active caspase 6\n<\/p>\n
(1:100, BioVision), Tuj1 (1:500, Covance), p75NTR (Chemicon), 2H3 (1:200,\n<\/p>\n
DSHB), Islet1\/2 (1:100, Santa Cruz Biotech), N-APP (polyclonal, 1:100, Thermo\n<\/p>\n
Fisher Scientific; monoclonal 22C11, Calbiochem), DR6 (R&D), NGF (Abcam\n<\/p>\n
and Genentech), BDNF (Calbiochem), NT3 (Genentech), amyloid-b (4G8,
\nCovance), the C-terminal cleavage-specific anti-amyloid-b antibody (anti-
\nAb(33\u201342), Sigma), and the C-terminal cleavage site of sAPPb (Covance).
\nMonoclonal antibodies to human DR6 ectodomain fused with human Fc\n<\/p>\n
(A.N., K. Dodge, V. Dixit and M.T.L., manuscript in preparation) were screened\n<\/p>\n
for binding to murine DR6 and block of commissural neuron degeneration.\n<\/p>\n
Proteins used were: netrin-1 (R&D), NGF (Roche), BDNF and NT3\n<\/p>\n
(Calbiochem), and control IgG (R&D). Transiently expressed murine DR6 ecto-\n<\/p>\n
domain (amino acids 1\u2013349) fused to human Fc, His-tagged human APP(1\u2013\n<\/p>\n
286), and APP(1\u2013286) and APLP2(1\u2013300) fused to human Fc, were affinity-\n<\/p>\n
purified from CHO cell supernatants (similar results were obtained using\n<\/p>\n
APP(1\u2013286) and APP(1\u2013306), Novus Biologicals). Inhibitors were used against:\n<\/p>\n
caspase 3 (Z-DEVD-FMK, Calbiochem), caspase 6 (Z-VEID-FMK, BD\n<\/p>\n
Pharmingen), BACE (OM99-2, Calbiochem; BACE inhibitor IV, Calbiochem;\n<\/p>\n
AZ29, Genentech), c-secretase (DAPT, Calbiochem).\n<\/p>\n
See Methods for details of in situ hybridization and immunohistochemistry,\n<\/p>\n
explant, dissociated, and Campenot chamber cultures, siRNA transfection,\n<\/p>\n
ARTICLES NATURE | Vol 457 | 19 February 2009\n<\/p>\n
988
\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n<\/div>\n
tracing and quantification of retinotectal projections, alkaline-phosphatase-
\nbinding assays and pull-down assays.\n<\/p>\n
Full Methods and any associated references are available in the online version of
\nthe paper at www.nature.com\/nature.\n<\/p>\n
Received 24 May; accepted 31 December 2008.\n<\/p>\n
1. Raff, M. C., Whitmore, A. V. & Finn, J. T. Axonal self-destruction and
\nneurodegeneration. Science 296, 868\u2013871 (2002).\n<\/p>\n
2. Luo, L. & O\u2019Leary, D. D. Axon retraction and degeneration in development and
\ndisease. Annu. Rev. Neurosci. 28, 127\u2013156 (2005).\n<\/p>\n
3. Buss, R. R., Sun, W. & Oppenheim, R. W. Adaptive roles of programmed cell death
\nduring nervous system development. Annu. Rev. Neurosci. 29, 1\u201335 (2006).\n<\/p>\n
4. Saxena, S. & Caroni, P. Mechanisms of axon degeneration: from development to
\ndisease. Prog. Neurobiol. 83, 174\u2013191 (2007).\n<\/p>\n
5. Haase, G., Pettmann, B., Raoul, C. & Henderson, C. E. Signaling by death receptors
\nin the nervous system. Curr. Opin. Neurobiol. 18, 284\u2013291 (2008).\n<\/p>\n
6. Kuida, K. et al. Decreased apoptosis in the brain and premature lethality in CPP32-
\ndeficient mice. Nature 384, 368\u2013372 (1996).\n<\/p>\n
7. White, F. A., Keller-Peck, C. R., Knudson, C. M., Korsmeyer, S. J. & Snider, W. D.
\nWidespread elimination of naturally occurring neuronal death in Bax-deficient
\nmice. J. Neurosci. 18, 1428\u20131439 (1998).\n<\/p>\n
8. Finn, J. T. et al. Evidence that Wallerian degeneration and localized axon
\ndegeneration induced by local neurotrophin deprivation do not involve caspases.
\nJ. Neurosci. 20, 1333\u20131341 (2000).\n<\/p>\n
9. Kuo, C. T., Zhu, S., Younger, S., Jan, L. Y. & Jan, Y. N. Identification of E2\/E3
\nubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron
\ndendrite pruning. Neuron 51, 283\u2013290 (2006).\n<\/p>\n
10. Williams, D. W., Kondo, S., Krzyzanowska, A., Hiromi, Y. & Truman, J. W. Local
\ncaspase activity directs engulfment of dendrites during pruning. Nature Neurosci.
\n9, 1234\u20131236 (2006).\n<\/p>\n
11. Plachta, N. et al. Identification of a lectin causing the degeneration of neuronal
\nprocesses using engineered embryonic stem cells. Nature Neurosci. 10, 712\u2013719
\n(2007).\n<\/p>\n
12. Sagot, Y. et al. Bcl-2 overexpression prevents motoneuron cell body loss but not
\naxonal degeneration in a mouse model of a neurodegenerative disease. J. Neurosci.
\n15, 7727\u20137733 (1995).\n<\/p>\n
13. Watts, R. J., Hoopfer, E. D. & Luo, L. Axon pruning during Drosophila
\nmetamorphosis: evidence for local degeneration and requirement of the
\nubiquitin-proteasome system. Neuron 38, 871\u2013885 (2003).\n<\/p>\n
14. Bodmer, J. L., Schneider, P. & Tschopp, J. The molecular architecture of the TNF
\nsuperfamily. Trends Biochem. Sci. 27, 19\u201326 (2002).\n<\/p>\n
15. Bossen, C. et al. Interactions of tumor necrosis factor (TNF) and TNF receptor
\nfamily members in the mouse and human. J. Biol. Chem. 281, 13964\u201313971
\n(2006).\n<\/p>\n
16. Pan, G. et al. Identification and functional characterization of DR6, a novel death
\ndomain-containing TNF receptor. FEBS Lett. 431, 351\u2013356 (1998).\n<\/p>\n
17. Zhao, H. et al. Impaired c-Jun amino terminal kinase activity and T cell
\ndifferentiation in death receptor 6-deficient mice. J. Exp. Med. 194, 1441\u20131448
\n(2001).\n<\/p>\n
18. Schmidt, C. S. et al. Enhanced B cell expansion, survival, and humoral responses by
\ntargeting death receptor 6. J. Exp. Med. 197, 51\u201362 (2003).\n<\/p>\n
19. Walsh, D. M. et al. The APP family of proteins: similarities and differences.
\nBiochem. Soc. Trans. 35, 416\u2013420 (2007).\n<\/p>\n
20. Reinhard, C., Hebert, S. S. & De Strooper, B. The amyloid-b precursor protein:
\nintegrating structure with biological function. EMBO J. 24, 3996\u20134006 (2005).\n<\/p>\n
21. Hardy, J. & Selkoe, D. J. The amyloid hypothesis of Alzheimer\u2019s disease: progress
\nand problems on the road to therapeutics. Science 297, 353\u2013356 (2002).\n<\/p>\n
22. Haass, C. & Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons
\nfrom the Alzheimer\u2019s amyloid b-peptide. Nature Rev. Mol. Cell Biol. 8, 101\u2013112
\n(2007).\n<\/p>\n
23. Wang, H. & Tessier-Lavigne, M. En passant neurotrophic action of an intermediate
\naxonal target in the developing mammalian CNS. Nature 401, 765\u2013769 (1999).\n<\/p>\n
24. Campenot, R. B., Walji, A. H. & Draker, D. D. Effects of sphingosine, staurosporine,
\nand phorbol ester on neurites of rat sympathetic neurons growing in
\ncompartmented cultures. J. Neurosci. 11, 1126\u20131139 (1991).\n<\/p>\n
25. Cole, S. L. & Vassar, R. BACE1 structure and function in health and Alzheimer\u2019s
\ndisease. Curr. Alzheimer Res. 5, 100\u2013120 (2008).\n<\/p>\n
26. Edwards, P. D. et al. Application of fragment-based lead generation to the
\ndiscovery of novel, cyclic amidine b-secretase inhibitors with nanomolar potency,
\ncellular activity, and high ligand efficiency. J. Med. Chem. 50, 5912\u20135925 (2007).\n<\/p>\n
27. Slunt, H. H. et al. Expression of a ubiquitous, cross-reactive homologue of the
\nmouse b-amyloid precursor protein (APP). J. Biol. Chem. 269, 2637\u20132644 (1994).\n<\/p>\n
28. Hilbich, C., Monning, U., Grund, C., Masters, C. L. & Beyreuther, K. Amyloid-like
\nproperties of peptides flanking the epitope of amyloid precursor protein-specific
\nmonoclonal antibody 22C11. J. Biol. Chem. 268, 26571\u201326577 (1993).\n<\/p>\n
29. Wang, P. et al. Defective neuromuscular synapses in mice lacking amyloid
\nprecursor protein (APP) and APP-like protein 2. J. Neurosci. 25, 1219\u20131225 (2005).\n<\/p>\n
30. Singh, K. K. et al. Developmental axon pruning mediated by BDNF-p75NTR-
\ndependent axon degeneration. Nature Neurosci. 11, 649\u2013658 (2008).\n<\/p>\n
31. Deppmann, C. D. et al. A model for neuronal competition during development.
\nScience 320, 369\u2013373 (2008).\n<\/p>\n
32. LeBlanc, A., Liu, H., Goodyer, C., Bergeron, C. & Hammond, J. Caspase-6 role in
\napoptosis of human neurons, amyloidogenesis, and Alzheimer\u2019s disease. J. Biol.
\nChem. 274, 23426\u201323436 (1999).\n<\/p>\n
33. Horowitz, P. M. et al. Early N-terminal changes and caspase-6 cleavage of tau in
\nAlzheimer\u2019s disease. J. Neurosci. 24, 7895\u20137902 (2004).\n<\/p>\n
34. Guo, H. et al. Active caspase-6 and caspase-6-cleaved tau in neuropil threads,
\nneuritic plaques, and neurofibrillary tangles of Alzheimer\u2019s disease. Am. J. Pathol.
\n165, 523\u2013531 (2004).\n<\/p>\n
35. Klaiman, G., Petzke, T. L., Hammond, J. & Leblanc, A. C. Targets of caspase-6
\nactivity in human neurons and Alzheimer disease. Mol. Cell. Proteomics 7,
\n1541\u20131555 (2008).\n<\/p>\n
36. Young-Pearse, T. L., Chen, A. C., Chang, R., Marquez, C. & Selkoe, D. J. Secreted
\nAPP regulates the function of full-length APP in neurite outgrowth through
\ninteraction with integrin b1. Neural Develop. 3, 15 (2008).\n<\/p>\n
37. Heber, S. et al. Mice with combined gene knock-outs reveal essential and partially
\nredundant functions of amyloid precursor protein family members. J. Neurosci. 20,
\n7951\u20137963 (2000).\n<\/p>\n
38. Perez, R. G., Zheng, H., Van der Ploeg, L. H. & Koo, E. H. The b-amyloid precursor
\nprotein of Alzheimer\u2019s disease enhances neuron viability and modulates neuronal
\npolarity. J. Neurosci. 17, 9407\u20139414 (1997).\n<\/p>\n
39. Han, P. et al. Suppression of cyclin-dependent kinase 5 activation by amyloid
\nprecursor protein: a novel excitoprotective mechanism involving modulation of
\nTau phosphorylation. J. Neurosci. 25, 11542\u201311552 (2005).\n<\/p>\n
40. Matrone, C. et al. Activation of the amyloidogenic route by NGF deprivation
\ninduces apoptotic death in PC12 cells. J. Alzheimers Dis. 13, 81\u201396 (2008).\n<\/p>\n
41. Matrone, C., Ciotti, M. T., Mercanti, D., Marolda, R. & Calissano, P. NGF and BDNF
\nsignaling control amyloidogenic route and Ab production in hippocampal
\nneurons. Proc. Natl Acad. Sci. USA 105, 13139\u201313144 (2008).\n<\/p>\n
42. Medana, I. M. & Esiri, M. M. Axonal damage: a key predictor of outcome in human
\nCNS diseases. Brain 126, 515\u2013530 (2003).\n<\/p>\n
43. Chiang, L. W. et al. An orchestrated gene expression component of neuronal
\nprogrammed cell death revealed by cDNA array analysis. Proc. Natl Acad. Sci. USA
\n98, 2814\u20132819 (2001).\n<\/p>\n
44. Muller, T., Meyer, H. E., Egensperger, R. & Marcus, K. The amyloid precursor
\nprotein intracellular domain (AICD) as modulator of gene expression, apoptosis,
\nand cytoskeletal dynamics-relevance for Alzheimer\u2019s disease. Prog. Neurobiol. 85,
\n393\u2013406 (2008).\n<\/p>\n
45. Van Gool, D., De Strooper, B., Van Leuven, F. & Dom, R. Amyloid precursor protein
\naccumulation in Lewy body dementia and Alzheimer\u2019s disease. Dementia 6,
\n63\u201368 (1995).\n<\/p>\n
46. Palmert, M. R. et al. Antisera to an amino-terminal peptide detect the amyloid
\nprotein precursor of Alzheimer\u2019s disease and recognize senile plaques. Biochem.
\nBiophys. Res. Commun. 156, 432\u2013437 (1988).\n<\/p>\n
47. Bertram, L. & Tanzi, R. E. Alzheimer\u2019s disease: one disorder, too many genes?
\nHum. Mol. Genet. 13, R135\u2013R141 (2004).\n<\/p>\n
48. Doyle, J. P. et al. Application of a translational profiling approach for the
\ncomparative analysis of CNS cell types. Cell 135, 749\u2013762 (2008).\n<\/p>\n
49. Albrecht, S. et al. Activation of caspase-6 in aging and mild cognitive impairment.
\nAm. J. Pathol. 170, 1200\u20131209 (2007).\n<\/p>\n
50. Graham, R. K. et al. Cleavage at the caspase-6 site is required for neuronal
\ndysfunction and degeneration due to mutant huntingtin. Cell 125, 1179\u20131191
\n(2006).\n<\/p>\n
Supplementary Information is linked to the online version of the paper at
\nwww.nature.com\/nature.\n<\/p>\n
Acknowledgements We thank R. Axel, C. Bargmann, B. de Strooper, V. Dixit,
\nC. Henderson, J. Lewcock, R. Scheller, R. Vassar, R. Watts, and members of the
\nM.T.-L. laboratory for helpful discussions and suggestions, and critical reading of
\nthe manuscript, and A. Bruce for making the diagrams. We thank P. Hass and
\nmembers of his laboratory (Genentech) for generation and purification of the DR6
\nectodomain and APP(1\u2013286), and W.-C. Liang and Y. Wu (Genentech) for binding
\nexperiments with purified proteins. Supported by Genentech (A.N. and M.T.-L.)
\nand National Eye Institute grant R01 EY07025 (D.D.M.O.\u2019L.).\n<\/p>\n
Author Contributions A.N. performed most of the experiments, with the exception
\nof the analysis of retinal projections and the experiments listed in the
\nAcknowledgements, and co-wrote the paper. The retinotectal analysis was
\nperformed by T.M. and supervised by D.D.M.O.\u2019L. M.T.-L. supervised or
\nco-supervised all experiments, and co-wrote the paper.\n<\/p>\n
Author Information Reprints and permissions information is available at NATURE | Vol 457 | 19 February 2009 ARTICLES\n<\/p>\n 989 METHODS 35 mRNA hybridization was as described51, using the mRNA locator kit (Ambion).\n<\/p>\n Radiolabelled mRNA probes to antisense sequences of mouse TNF receptor\n<\/p>\n superfamily member 39 untranslated regions were generated using the\n<\/p>\n MAXIscript kit (Ambion). Immunochemistry was as described on sections51\n<\/p>\n or cultured cells52. Surface labelling was done without detergent. Double label-\n<\/p>\n ling was performed using Zenon Technology (Invitrogen). Fluorescent caspase recommended (in situ cell death detection kit, Roche).\n<\/p>\n Quantification of axon degeneration. To measure the percentage of degenerat- counted.\n<\/p>\n Quantification on sections. Twenty-micrometre serial cryosections were taken 2\/2 zygous littermates. Motor neurons were counted in all sections at E14.5 (large\n<\/p>\n Islet1\/2-positive ventral neurons; 4 mutants, 3 controls) or E18 (large H&E-\n<\/p>\n stained ventral neurons; 7 mutants, 5 controls).\n<\/p>\n Neuronal cultures. E13 rat dorsal spinal cord was dissected after the introduc- assay was as described23. DR6 siRNA1 and siRNA2 (sense) were 59-CAAU-\n<\/p>\n AGGUCAGGAAGAUGGCU-39 and 59-AAUCUGUUGAGUUCAUGCCUU-39,\n<\/p>\n respectively. The mismatch sequence complementary to siRNA1 was 59- and motor neuron explants or dissociated cells were cultured on laminin-coated\n<\/p>\n 35-mm tissue culture dishes in culture medium (Neurobasal medium with B27\n<\/p>\n supplement) with appropriate trophic factor (sensory: NGF, 50 mg ml BDNF and NT3, 10 mg ml growth factor and adding appropriate antibodies (sensory: anti-NGF,\n<\/p>\n 50 mg ml 21 of siRNAs into dissociated sensory neuron cultures was performed as\n<\/p>\n described54.\n<\/p>\n Campenot chamber assay. The Campenot chamber assay was carried out as coated with poly-D-lysine and laminin and scratched with a pin rake (Tyler\n<\/p>\n Research) to generate tracks, as illustrated in Fig. 2a. A drop of culture medium\n<\/p>\n containing 4 mg ml methylcellulose was placed on the scratched substratum. A\n<\/p>\n teflon divider (Tyler Research) was seated on silicone grease and a dab of silicone\n<\/p>\n grease was placed at the mouth of the centre slot. Dissociated sensory neurons\n<\/p>\n from E12.5 mouse DRGs were suspended in methylcellulose-containing med- injected into the centre slot under a dissecting microscope, and allowed to settle\n<\/p>\n overnight. The outer perimeter of the dish (the cell body compartment) and the\n<\/p>\n inner axonal compartments were filled with methylcellulose-containing med-\n<\/p>\n ium. Within 3\u20135 days in vitro, axons begin to emerge into left and right compart-\n<\/p>\n ments. To trigger local axonal degeneration, NGF-containing medium from\n<\/p>\n axonal compartments was replaced with neurobasal medium containing anti-\n<\/p>\n NGF. Where indicated, anti-DR6.1 or control IgG were added (50 mg ml final\n<\/p>\n concentration) at the time of NGF deprivation. Cultures were fixed at different\n<\/p>\n times after deprivation with 4% paraformaldehyde for 30 min at room temper-\n<\/p>\n ature and processed for TuJ1 immunofluorescence.\n<\/p>\n Tracing of RGC axons. Injections of DiI into temporal retina and subsequent termination zone was determined to be the centre of a circumscribed circle.\n<\/p>\n Injection size, termination zone size and the efficiency of axon labelling were\n<\/p>\n not different between wild-type and mutant. The termination zone position was\n<\/p>\n also unchanged (average termination zone centre: 50.3% for controls and 49.8% morphologically normal, with all retinal layers present in similar proportions to\n<\/p>\n wild-type.\n<\/p>\n For quantification, the presence of axons was determined in 100-mm vibra- graphed and axon presence was recorded in 100-mm segments from the anterior edge of the superior colliculus, these data were transposed from photos of sec-\n<\/p>\n tions to photos of the wholemount superior colliculuses in dorsal view, resulting\n<\/p>\n in a grid of 100-mm squares covering each superior colliculus. The termination blinded to genotype.\n<\/p>\n Alkaline phosphatase binding assays. Alkaline phosphatase fused to the DR6 expressed in COS-1 cells. Medium was changed after 12 h to OPTI-MEM The DR6\u2013AP blot assay on conditioned medium was performed as\n<\/p>\n described56. In brief, conditioned medium derived from sensory axons main-\n<\/p>\n tained in Campenot chambers or ventral spinal cord explants in explant culture\n<\/p>\n (with or without trophic deprivation) was concentrated tenfold using centriprep\n<\/p>\n centrifugal filters (Millipore), resolved in 4\u201320% gels under non-reducing con-\n<\/p>\n ditions, and blotted with DR6\u2013AP in alkaline phosphatase binding buffer.\n<\/p>\n For in situ sensory axon binding assays, wild-type or Bax sensory explants\n<\/p>\n were cultured, deprived of NGF for 12 h (with or without BAX inhibitor, as\n<\/p>\n indicated), then washed twice with the alkaline phosphatase binding buffer\n<\/p>\n (HBSS, Gibco, with 0.2% BSA, 0.1% NaN3, 5 mM CaCl2, 1 mM MgCl2,\n<\/p>\n 20 mM HEPES, pH 7.0). The alkaline phosphatase binding assay was carried\n<\/p>\n out by making a 1:1 mixture of binding buffer and conditioned medium contain-\n<\/p>\n ing DR6\u2013AP, APP\u2013AP, or control alkaline phosphatase, applied to DRG explants\n<\/p>\n in 8-well culture slides and incubated for 90 min at room temperature. Explants\n<\/p>\n were rinsed five times with binding buffer, fixed with formaldehyde (3.7% in\n<\/p>\n PBS) for 12 min at room temperature, and rinsed three times with HBS (20 mM\n<\/p>\n HEPES, pH 7.0, 150 mM NaCl). Endogenous alkaline phosphatase activity was 50 mM MgCl2), bound alkaline phosphatase fusion was visualized by developing\n<\/p>\n colour stain in alkaline phosphatase reaction buffer with 1\/50 (by volume) of\n<\/p>\n NBT\/BCIP stock solution (Roche) overnight at room temperature.\n<\/p>\n The in situ binding of DR6\u2013AP to COS cells transiently expressing APP695 was\n<\/p>\n performed in the same way, but with heparin (2\u201310 mg ml nonspecific binding. DR6\u2013AP did not bind to controls (p75NTR or DR6\n<\/p>\n expressed in COS-1 cells) under these conditions. For quantitative analysis,\n<\/p>\n the amount of DR6\u2013AP protein in medium was quantified as described57. In\n<\/p>\n brief, 100 ml of 23 alkaline phosphatase buffer (prepared by adding 100 mg para- lamine, pH 9.8) was mixed with conditioned medium containing DR6\u2013AP or\n<\/p>\n control alkaline phosphatase. The reaction was developed over 5\u201315 min, with\n<\/p>\n the absorbance being in the linear range (0.1\u20131). The volume of reaction was\n<\/p>\n adjusted by adding 800 ml of distilled water, and absorbance was measured at 100ml): C (nM)5A31003(60\/developing time)\/30. Saturation binding analysis saturation binding curves and to determine half-maximal saturation value.\n<\/p>\n APP pull-down assays. Soluble ectodomains of TNF receptor superfamily mem- were incubated at 5 mg ml in binding buffer (HBSS, with 0.2% BSA, 0.1%\n<\/p>\n NaN3, 5 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, pH 7.0) with 1 mg ml of\n<\/p>\n His-tagged APP(1\u2013286) and protein A\/G beads (Santa Cruz Bio) at 4 uC over- eluted from beads with SDS loading buffer, resolved in 4\u201320% SDS PAGE gels,\n<\/p>\n and blotted for APP (with anti-NAPP(poly)) and for TNF receptor family mem-\n<\/p>\n bers (with anti-human Fc).\n<\/p>\n Mice. The following mutant mice were used: DR6 knockout17 (gift from V. (Jackson laboratory).\n<\/p>\n 51. Sabatier, C. et al. The divergent Robo family protein rig-1\/Robo3 is a negative 52. Atwal, J. K. et al. PirB is a functional receptor for myelin inhibitors of axonal 53. Chen, Z. et al. Alternative splicing of the Robo3 axon guidance Receptor governs 54. Higuchi, H., Yamashita, T., Yoshikawa, H. & Tohyama, M. Functional inhibition of 55. McLaughlin, T., Torborg, C. L., Feller, M. B. & O\u2019Leary, D. D. Retinotopic map 56. Pettmann, B. et al. Biological activities of nerve growth factor bound to 57. Okada, A. et al. Boc is a receptor for sonic hedgehog in the guidance of 58. Knudson, C. M. et al. Bax-deficient mice with lymphoid hyperplasia and male germ 59. Lee, K. F. et al. Targeted mutation of the gene encoding the low affinity NGF doi:10.1038\/nature07767\n<\/p>\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n
\nwww.nature.com\/reprints. The authors declare competing financial interests:
\ndetails accompany the full-text HTML version of the paper at www.nature.com\/
\nnature. Correspondence and requests for materials should be addressed to M.T.-L.
\n([email\u00a0protected]<\/a>).\n<\/p>\n
\n Macmillan Publishers Limited. All rights reserved\u00a92009<\/p>\n<\/p>\n
\nIn situ hybridization and immunochemistry. Radioactively labelled\n<\/p>\n
\nS in situ\n<\/p>\n
\nreporter assays were as recommended (MP Biologicals). TUNEL assays was as\n<\/p>\n
\ning axon bundles, the number of still visible bundles that showed breakdown was\n<\/p>\n
\nfrom axially matched cervical (C1\u2013C3) levels of DR6\n<\/p>\n
\nembryos and hetero-\n<\/p>\n
\ntion of plasmids and siRNAs by electroporation53; the dorsal explant survival\n<\/p>\n
\nGGACTCTGTGTACAGTCACCTCCCAGATCTGTTATAG-39. Mouse sensory\n<\/p>\n
\n21
\n; motor:\n<\/p>\n
\n21
\n). Trophic deprivation was achieved by removing\n<\/p>\n
\n21
\n; motor: anti-BDNF and anti-NT3, 50 mg ml\n<\/p>\n
\n). The introduction\n<\/p>\n
\ndescribed24 with minor modifications. In brief, 35-mm tissue culture dishes were\n<\/p>\n
\n21\n<\/p>\n
\nium, loaded into a disposable sterile syringe fitted with a 22-gauge needle,\n<\/p>\n
\n21\n<\/p>\n
\nanalyses were performed essentially as previously described55. The centre of the\n<\/p>\n
\nfor mutants (P . 0.9), from the medial edge). The retina in mutants appeared\n<\/p>\n
\ntome sections and transposed onto the superior colliculus. Sections were photo-\n<\/p>\n
\nborder. Using landmarks such as the termination zone, unique arbors, or the\n<\/p>\n
\nzones and grids were aligned for the analyses. All analyses were performed\n<\/p>\n
\nectodomain (DR6\u2013AP) and to APP(1\u2013286) (APP\u2013AP) were transiently\n<\/p>\n
\n(Invitrogen), and conditioned medium was collected 36 h later and filtered.\n<\/p>\n
\n2\/2\n<\/p>\n
\nblocked by heat inactivation at 65 uC in HBS for 30 min. After rinsing three times
\nin alkaline phosphatase reaction buffer (100 mM Tris, pH 9.5, 100 mM NaCl,\n<\/p>\n
\n21
\n) added to reduce\n<\/p>\n
\nnitrophenyl phosphate (Sigma) and 15 ml of 1 M MgCl2 to 15 ml 2 M diethano-\n<\/p>\n
\n405 nm. Concentration in nM was calculated according to the formula (for\n<\/p>\n
\nwas performed as described57. Prizm 4 software (GraphPad) was used to generate\n<\/p>\n
\nbers fused to human Fc were expressed in CHO cells and affinity purified. They\n<\/p>\n
\n21\n<\/p>\n
\n21\n<\/p>\n
\nnight. Beads were washed five times with binding buffer. Bound complexes were\n<\/p>\n
\nDixit), Bax knockout58 (gift from S. Korsmeyer) and p75NTR knockout59\n<\/p>\n
\nregulator of slit responsiveness required for midline crossing by commissural
\naxons. Cell 117, 157\u2013169 (2004).\n<\/p>\n
\nregeneration. Science 322, 967\u2013970 (2008).\n<\/p>\n
\nthe midline switch. Neuron 58, 325\u2013332 (2008).\n<\/p>\n
\nthe p75 receptor using a small interfering RNA. Biochem. Biophys. Res. Commun.
\n301, 804\u2013809 (2003).\n<\/p>\n
\nrefinement requires spontaneous retinal waves during a brief critical period of
\ndevelopment. Neuron 40, 1147\u20131160 (2003).\n<\/p>\n
\nnitrocellulose paper by western blotting. J. Neurosci. 8, 3624\u20133632 (1988).\n<\/p>\n
\ncommissural axons. Nature 444, 369\u2013373 (2006).\n<\/p>\n
\ncell death. Science 270, 96\u201399 (1995).\n<\/p>\n
\nreceptor p75 leads to deficits in the peripheral sensory nervous system. Cell 69,
\n737\u2013749 (1992).\n<\/p>\n