Neuroscience Letters 372 (2004) 4651

TRAIL-related death receptors in normal, Lurcher

and weaver mutant mouse brain
Jorg Baurle , Sabine Frischmuth, Karel Kranda
Department of Physiology, ChariteUniversitatsmedizin Berlin, Campus Benjamin Franklin, Arnimallee 22, D-14195 Berlin, Germany
Received 8 July 2004; received in revised form 2 September 2004; accepted 3 September 2004

Abstract
In this study, we searched for murine analogues of the four death-receptor types (TRAIL-R1 to R4), targeted by the tumour necrosis factor
related apoptosis inducing ligand (TRAIL), which were recently identified in the human brain. The expression of TRAIL-receptors in the
normal murine brain was investigated using antibodies directed against different epitopes of the human TRAIL-receptors. Mouse mutants, in
particular weaver and Lurcher with their well defined spatio-temporal patterns of neurodegeneration in the cerebellum, the inferior olive and
the substantia nigra, were used as a model for investigating a potential contribution of TRAIL-receptors to the genetically determined cell
death observed in these mutants.
Although all antibodies used, recognized the respective human antigens, only the murine analogue of the human TRAIL-R2 epitope was
also identified in the mouse brain. Antisera against human TRAIL-R1, TRAIL-R3 and TRAIL-R4 failed to reveal any other murine TRAILreceptor analogue. In normal mice, TRAIL-R2 is not universally expressed throughout the brain but rather restricted to specific neuronal
populations predominantly consisting of large neurons.
In weaver, the spatial patterns and relative densities of TRAIL-R2 labelling were virtually identical to those seen in wild-types during the
period of cell death in the cerebellum and the substantia nigra. In Lurcher, TRAIL-R2 expression in cerebellar granule cells and inferior olivary
neurons was identical to that in wildtypes but significantly reduced in Purkinje cells undergoing degeneration. Thus, although TRAIL-R2 is
found to be expressed in various cell types of the murine brain, cell death in weaver and Lurcher mutants is apparently not accompanied by
an upregulation of TRAIL-receptors.
2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: TRAIL-R2; Mouse brain; Neurodegeneration; Apoptosis; Purkinje cells; Substantia nigra

Death receptors are cell surface molecules, which induce

apoptosis by activating intrinsic apoptotic cascades via an
intracellular death domain (DD). Tumour necrosis factor
(TNF) related apoptosis inducing ligand receptors (TRAILR) [11,22] are members of the TNF receptor gene superfamily
of cytokines [19]. Although four types of membrane-bound
TRAIL-receptors have been identified in humans to date, only
TRAIL-R1 (DR4) and TRAIL-R2 (DR5/Killer) actually possess a functional cytoplasmic DD [2,9,14,23]. The DD is
absent in TRAIL-R3 (DcR1) and so severely truncated in
TRAIL-R4 (DcR2) that its apoptotic potential is eliminated.
Whether TRAIL-R3 and TRAIL-R4 actually act as decoy re

Corresponding author. Tel.: +49 30 8445 1675; fax: +49 30 8445 1602.
E-mail address: baeurle@zedat.fu-berlin.de (J. Baurle).

0304-3940/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2004.09.009

ceptors, which compete with TRAIL-R1 and TRAIL-R2 for

the TRAIL ligand and thus protecting cells from apoptosis
[5,9,18] remains to be established.
Until recently, TRAIL-induced apoptosis has been almost
exclusively investigated in tumour cells [11,12,21,22], even
though TRAIL and its receptors are also expressed in normal tissue [11,22]. Hence, TRAIL may also induce apoptosis of neurons and glia [7,8]. If TRAIL can induce apoptosis
in the brain, then the presence of its receptors in the target
cells would be a precondition. However, the evidence for
this hypothesis is rather inconsistent. Although transcripts of
TRAIL-R1, TRAIL-R2 and TRAIL-R3 were found in normal
human brain cells [4] and all four death-receptor types could
be labelled in human neurons and glia [3], other investigation
reports the absence of TRAIL-R2 transcripts [18].

J. Baurle et al. / Neuroscience Letters 372 (2004) 4651

Recently, however, there have been reports of a possible

murine homologue to the human TRAIL-R2 with a functional DD, variously termed as MK [24] or p54mTRAIL-R
[6], and also two new murine TRAIL-decoy-receptors, named
mDcTRAIL-R1 and mDc TRAIL-R2 [15]. However, the expression and distribution of these receptors in the brain remains unknown.
The objective of this study was: (1) to examine whether
homologues to the human TRAIL-Rs are in principle expressed on a translational level in the murine brain and (2) to
search for a possible differential expression of these receptors in space (i.e. in various neuronal populations) and also
in time (postnatal age).
Furthermore, should TRAIL-induced apoptosis be mediated by a differential expression of TRAIL-Rs, then this study
may reveal the possible involvement of TRAIL-receptors in
apoptotic processes proceeding in mice mutants. In this study
we opted for the mutants weaver (wv/wv) and Lurcher (Lc/+)
as models for neurodegeneration, because the well-defined
gene defects of these mutants lead to the substantial death of
different neuronal populations with known spatio-temporal
patterns.
Wild-types (B6CBA) as well as Lc/+ and wv/wv mutant
mice, were the offspring of breeder pairs originally purchased
from Jackson Laboratories (Bar Harbor, USA). The animals
were kept under a normal dark/light cycle with food and water
ad lib. In total we investigated 44 murine brains (15 +/+; 15
Lc/+; 14 wv/wv) at postntal day (P) 6 (wv/wv only), P12, P15,
P19, P28, P60, P120, P240 and P364. Lc/+ mutants, aged
between P12 and 52 weeks, were phenotypically recognized
by their ataxia and histologically confirmed by their atrophic
cerebellum. Homozygous wv/wv offspring, aged between P6
and 52 weeks, originated from homozygous breeder pairs of
the wv/wv colony, kept at our institute. All experiments were
approved by the local Animal Care and Use Committee and
conform to NIH guidelines.
After a lethal dose (1.75 g/kg body weight) of chloral hydrate, mice were transcardially perfused with saline at 37 C,
then with 1% paraformaldehyde and 1% glutaraldehyde in
0.1 M PO4 buffer at pH 7.4 and 4 C for 30 min. Brains were
immediately dissected, embedded in agar and cut in ice-cold
PO4 buffer with a vibratome (Series 1000; TPI Inc. St. Louis)
at 50 m intervals. We opted for the above mixture as it appeared to be preferable (better mechanical properties of the
fixed brains and lower immunohistochemical background) to
the use of 1% or 4% paraformaldehyde, which we tested and
evaluated in parallel on several brains. The different fixatives
did not affect the staining characteristics of the different antibodies. Consecutive sections were immediately processed
for anti-TRAIL-R using free floating immunohistochemistry.
To determine whether dopaminergic neurons of the substantia nigra express TRAIL-R, adjacent sections of the mesencephalon were immunoreacted for tyrosine hydroxylase
(TH).
TRAIL-R1: (1) Polyclonal rabbit anti-DR4 (TRAIL-R1;
No. 1139, ProSci), raised against a peptide corresponding

47

to amino acids (aa) 427445 of human DR4 mature protein (1:200). (2) Monoclonal mouse anti-TRAIL-R1 (Alexis,
ALX-804-297A, 1:100) against recombinant TRAIL-R1:Fc.
(3) The extracellular domain (aa 24239) of human TRAILR1 (ALX-522-004), fused to the Fc portion of human IgG1.
TRAIL-R2: (1) Polyclonal rabbit anti-DR5 (TRAIL-R2;
AB16942, Chemicon), raised against a peptide corresponding
to aa 388407 of the human DR5 precursor (1:200). (2) aa
388407 of human DR5 (Chemicon) for pre-absorption of
anti-TRAIL-R2.
TRAIL-R3: (1) Polyclonal goat anti-TRAIL-R3 (ALX210-744), raised against a synthetic peptide corresponding to
aa 73103 of the extracellular domain of human TRAIL-R3
(1:200). (2) Monoclonal mouse anti human TRAIL-R3:Fc
(ALX-804-344A). (3) The extracellular domain (aa 25240)
of human TRAIL-R3 (ALX-522-006), fused to the FC portion of human IgG1.
TRAIL-R4: Polyclonal rabbit anti-DcR2 (TRAIL-R4; AB
16943, Chemicon), directed against aa 249263 of the human
DcR2 precursor (1:200).
Polyclonal rabbit anti-TH (9360-0004; Biotrend), raised
against native rat TH (1:1000).
Immunohistochemistry followed a standard protocol of
the peroxidase-antiperoxidase (PAP)-method [20] using unconjugated secondary antibodies (Dako) at a dilution of 1:100
for 2 h. After 1 h incubation with the PAP-complex (1:50;
Dako) the binding sites were visualised by reacting peroxidase with 0.05% diaminobenzidine (DAB, Sigma) in Tris
buffered saline containing 0.03% H2 O2 for 5 min. Consecutive sections were also incubated with biotinylated or HRPconjugated secondary antibodies (Dako), respectively, to test
for possible method specific variation. Although all different
methods identified the same neuronal populations in the different murine strains, the PAP-method produced the lowest
unspecific background labelling. Age matched mutant and
wild-type brains were incubated and processed simultaneously in the same solutions. Omission of primary antisera
resulted in a complete lack of immunostaining in all cases
(see also Fig. 2A).
Dot blots, western blotting and pre-absorption: Cell
lysates of human HeLa and Jurkat DC+ cells, as well as cerebellar homogenates from normal, wv/wv and Lc/+ mice were
either spotted onto nitrocellulose (Protran BA 85, Schleicher
& Schuell) in a volume of about 0.2 l (equivalent to about
0.35 g protein; Lowry method) or subjected to SDSPAGE
electrophoresis on 12.5% polyacrylamide gels and blotted
(25 g; Lowry method) according to standard protocols. Dot
blots were immunoreacted together with brain sections of
age-matched mice. Immunodetection on western blots was
based on chemiluminescence (west femto substrate; Pierce,
USA). Prior to immunoincubation of blots and tissue sections
the primary antibodies were in several instances pre-absorbed
with their respective immunogen at molecular ratios of 1:3.
Purkinje cells (PC) and substantia nigra pars compacta (SNpc) neurons of wild-types and mutants were randomly sampled from TRAIL-R2-immunoreacted sections

48

J. Baurle et al. / Neuroscience Letters 372 (2004) 4651

Fig. 1. Specificity testing. (A) Dot blots immunoreacted for anti-TRAILR2. The left blot represents the not pre-absorbed condition, whereas the
right blot shows the pre-absorbed condition, where the blocking peptide
was added before the incubation. (B) The left hemisection from wv/wv was
pre-absorbed with the control peptide before immunoincubation with antiTRAIL-R2, whereas the right hemisection from +/+ was not pre-absorbed.
(C) Western blot with anti-TRAIL-R2 identifying 60 kDa Proteins in HeLa
whole cell lysates, as well as in the brain homogenates of normal (+/+)
and mutant mice (wv/wv and Lc/+). (D) Western blot with anti-TRAIL-R4
identifying a 35 kDa Protein in HeLa whole cell lysates (the trace on the
left). This protein could not be identified in the brain homogenates of normal
(+/+) and mutant mice (wv/wv and Lc/+) as documented by the absence of
any trace.

(minimum 10 sections/animal) of age-matched mice (three

animals for each genotype) and analysed densitometrically
using a 40 objective (Zeiss Ultraphot, Germany), a Videocamera (Hitachi HV-C20) and the Neurolucida software
(Ver. 4.36; MicroBrightField). Somata of individual neurons were outlined and the integral luminance inside the
contour was matched against a reference area containing
no tissue (brightness reference value = 165). The Wilcoxon
(MannWhitney U-test) test for independent samples was
employed for statistical evaluation. Note, that one limiting
factor for accurate quantitative evaluation of the relative antigen concentration from the measured densitometric values is
the potentially nonlinear relationship of these two variables.

TRAIL-receptors are known to be expressed abundantly

in human HeLa and Jurkat cells. To ascertain whether the
antibodies used in this study could recognize the human antigens, HeLa and Jurkat whole cell lysates were immunoincubated together with cerebellar homogenates of wild-types
and wv/wv as well as Lc/+ mutants in dot blot and western blot experiments. All six different TRAIL-R antibodies,
used in this study, produced positive results in HeLa and/or
Jurkat cell lysates but only the TRAIL-R2 antibody yielded
immunopositivity also in the murine homogenates (see Fig. 1
for representative results with anti-TRAIL-R2 (AB 16942)
and anti-TRAIL-R4 (AB 16943), not shown for anti-TRAILR1 and R3). The left blot in Fig. 1A (the not pre-absorbed
condition) shows specific reaction for anti-TRAIL-R2 with aa
388407 of human DR5 precursor (control peptide) and HeLa
and Jurkat cell lysates as well as normal and mutant mouse
brain. The right blot (the pre-absorbed condition) shows that
pre-absorption with the immunogen of the primary antibody
almost completely prevented the staining of the samples. The
central dot labelled with anti-TRAIL-R2 indicates that, apart
from the primary antiserum, the immunodetection procedure
was adequate in both conditions.
Furthermore, pre-absorption of the primary antisera with
the appropriate immunogens (peptides) prevented immunostaining not only of the human and murine homogenates in
the dot and western blots but also in mouse brain sections
(Fig. 1B). Note the virtually complete absence of immunolabelling in the left and the clear delineation of the PC-layer by
immunopositve PC in the right hemisection. Fig. 1C shows
three visible bands in the vicinity of 60 kDa, all of which disappear after pre-absorption of the antisera (not shown) with
the appropriate antigen.
Immunolocalisation of amino acid sequences, related to
human TRAIL-R1 through TRAIL-R4 or to their precursors, showed that apart from the targeted epitope recognized
by anti-TRAIL-R2, none of the other human TRAIL-R1,
TRAIL-R3 and TRAIL-R4 epitopes could be identified in
the murine brains (Figs. 1 and 2). The analysis of brain sections revealed that TRAIL-R2 is not ubiquitous in murine
brains but rather restricted to specific neuronal populations
and brain structures (Fig. 2). This pattern was observed in
animals of all ages studied (P6 until P364).
Prominent labelling appeared predominantly in large
neurons of the cerebellar, vestibular and cochlear nuclei
(Fig. 2CF), the substantia nigra (Fig. 2G), the trapezoid
body (Fig. 2I) and especially in the locus coeruleus (Fig. 2H).
The entorhinal cortex (Fig. 2J) was the most prominently labelled cortical structure. In the cerebellar cortex (Fig. 2B,
DF) virtually all PC were immunopositive as opposed to
only a few immunolabeled Golgi cells (Fig. 2D) and the absence of immunostaining in the granule cell population. Although labelled dendrites of neurons in the substantia nigra
were relatively frequent (Fig. 2G), we are yet to discover any
single labelled dendrite of PC in the cerebellum (Fig. 2DF).
As in wild-types, immunopositivity in wv/wv and Lc/+
was seen only after immunoincubation with anti-TRAIL-R2

J. Baurle et al. / Neuroscience Letters 372 (2004) 4651

49

Fig. 2. Distribution of TRAIL-R2 immunoreactivity in the brain of wild-types and cerebellar mutants. (A) The absence of primary antiserum (control condition).
(B) Anti-TRAIL-R2 immunoreacted section from a 15-day-old wild-type sectioned at the same rostrocaudal level as shown in A. (C) Higher magnification of
the deep cerebellar and vestibular nuclei in a 19-day-old wv/wv mutant. (D) Cerebellar cortex of a 15-day-old wild-type. PC (white arrows) and a single Golgi
cell (GoC, black arrow) in the granular layer (GL) are immunopositive. (E) Ectopically located PCs (white arrows) of a 15-day-old wv/wv mutant. (F) PC (white
arrows) of a 15-day-old Lc/+ mutant. (G) Labeled neurons in the SNpc of a 12-month-old Lc/+ mutant. (H) Dense labelling of neurons (arrows) in the locus
coeruleus of a 12-month-old wild-type. (I) Trapezoid body of a 19-day-old wv/wv mutant. (J) TRAIL-R2 in the entorhinal cortex. Abbreviations: DL, dissecant
lamina; GL, granular layer; GoC, Golgi cell; ML, molecular layer; MNTB, medial nucleus of the trapezoid body; PC, Purkinje cell; PEL, principal external
lamina; PIL, principal internal lamina; PL, plexiform lamina; VNTB, ventral nucleus of the trapezoid body. Calibration bars: A, also valid for B: 1000 m; C:
300 m; D, also valid for E, F: 100 m; G: 100 m; H: 100 m; I, also valid for J: 100 m.

but not with antibodies raised against the other TRAIL-R.

Furthermore, in both mutant types, the neuronal populations
actually labelled by anti-TRAIL-R2 were identical to those
seen in +/+. This pattern was also observed for cerebellar
granule cells (GC) and inferior olivary (IO) neurons. These
neurons remained immunonegative in +/+ as well as in wv/wv
and Lc/+, even though GC undergo postnatal degeneration in
both mutants and IO neurons die in Lc/+.
Apart from GC, about 25% of PC and the majority of
dopaminergic neurons in the SNpc of wv/wv degenerate postnatally. These neuronal types are TRAIL-R2 immunopositive
in +/+ as well as in the mutants (Fig. 2DG), but an enhanced
TRAIL-R2 labelling, which could be considered as a sign of
apoptotic upregulation, was not observed in wv/wv (Table 1).

Although virtually all PC are lost during the first 34 postnatal weeks in Lc/+, TRAIL-R2 labelling of dying PC is paradoxically lower than seen in wild-types or wv/wv (Fig. 2DF
and Table 1). Note the almost identical brightness of PC in
+/+ and wv/wv (Table 1; level of significance: p = 0.589;
MannWhitney U-test). The brightness measured in Lc/+ PC
is significantly (p < 0.0005) higher than in wild-types, hence
reflecting a lower staining density. In the SNpc, no significant
density differences were detected between +/+ and wv/wv (p
= 0.37) or +/+ and Lc/+ (p = 0.26).
Here we present for the first time evidence for the expression of TRAIL-R2 on a translational level in the mouse
brain and demonstrate that the distribution of these receptors is restricted to predominantly large neurons, localised in

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J. Baurle et al. / Neuroscience Letters 372 (2004) 4651

Table 1
Mean brightness and standard deviation values of anti-TRAIL-R2 immunoreacted Purkinje cells (PC) and neurons of the substantia nigra pars
compacta (SNpc) at P15
+/+

wv/wv

Lc/+

PC (n)
Mean brightness

249
124.3 (6.4)

251
125.2 (7.3)

251
137.9 (7.4)

SNpc (n)
Mean brightness

242
108.2 (10.1)

257
108.2 (7.4)

258
109.1 (11)

Brightness reference value = 165. n = number of individual neurons measured

in three animals of each genotype.

rather few brain regions. The absence of other human TRAILR analogues in the murine brain is consistent with results
from molecular cloning and biochemical experiments, describing only one murine homologue to the currently known
human TRAIL-R [6,24]. Antibodies directed against epitopes
of the human decoy receptors TRAIL-R3 and TRAIL-R4
failed to detect the recently described murine decoy receptors mDcTRAIL-R1 and mDcTRAIL-R2 [15] in the brain.
The absence of stained cells may reflect the low degree of sequence homology between the human and the murine decoy
receptors [15] rather than their absence in the brain.
As TRAIL-R2 is considered a functional death receptor
with an apoptosis-inducing capability [2,9,14,23], its sheer
presence in the mouse brain may indicate its participation in
inducing and regulating neuronal death. The appearance of
three bands (see Fig. 1C) rather than just the two described
in human as TRICK2A and TRICK2B [16], open the possibility that there is an additional not yet described murine
isoform. The selective expression of TRAIL-R2 in only a
few neuronal populations further suggests a particular role in
the apoptotic processes of these cells. Hence, a potential role
of TRAIL-R2 in regulating death in these specific neurons
may be revealed in those animal models where these cell
populations perish during primary and transsynaptic apoptotic processes. In wv/wv, GC, PC and SNpc neurons undergo postnatal degeneration [13] and in Lc/+, massive cell
loss occurs in the PC-, GC- and IO-populations [1,17]. From
this study, PC, as well as SNpc neurons are both TRAIL-R2
immunopositive in the normal mouse brain, while GC and
IO neurons do not express TRAIL-R2 in wild-types. Yet, the
results from wv/wv and Lc/+ mutants are not supportive of
a role for TRAIL-R2 in the cell death of either PC or SNpc
nor GC and IO neurons. Although the apoptotic nature of GC
death in wv/wv is indicated by caspase activation in these neurons [10], cerebellar GC of wv/wv remain immunonegative
for TRAIL-R2, even at the peak of neurodegeneration. Similarly, the labelling patterns and staining intensities of PC and
neurons in the SNpc of the wild-type are not different from
those in wv/wv. These observations seem incompatible with a
possible role of TRAIL-R2 in regulating the primary as well
as the trans-synaptic cell death in wv/wv mice.
Although active caspase-3 is found in dying PC, GC and
IO neurons of Lc/+ mutants [17], TRAIL-R2 labelling was
also absent in the GC-layer and in the IO of Lc/+ at all stages

of neurodegeneration. Paradoxically, the TRAIL-R2 staining intensity of PC in Lc/+ was even lower than in +/+ and
wv/wv, indicating a reduced, rather than enhanced TRAIL-R2
expression in dying PC of Lc/+. This low level of TRAIL-R2
in Lc/+ PC may reflect a possible functional and structural
decline of PC prior to their death rather than a downregulation
of receptor density.
Although TRAIL-induced apoptosis could presumably be
involved in age-related neuronal loss, the differential spatial
distribution of TRAIL-R2 throughout the murine brain is apparently invariant to the age of the mice. The anisotropic
TRAIL-R2 expression in the mouse brain may thus reflect
intrinsic neuronal features unrelated to the momentary apoptotic state in wv/wv or Lc/+. Furthermore, as spatial receptor distributions remain invariant over the lifespan examined,
TRAIL-R2 expression does not mirror developmental or agerelated cell loss.
Conceivably, TRAIL-induced cell death may be regulated
by mechanisms, which do not alter the expression and/or
distribution of TRAIL-receptors.

Acknowledgements
We wish to thank Miss H. Wolynski for expert technical
assistance and Miss G. Beyer for donating the human cell
lines. This study was supported in part by the Sonnenfeld
Stiftung.

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