The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells.

Abstract

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP (C) is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.

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www.landesbioscience.com Prion 1
ReseaRch PaPeR
ReseaRch PaPeR
Prion 6:3, 1-1; July/august 2012; © 2012 Landes Bioscience
The role of Bax and caspase-3 in doppel-induced
apoptosis of cerebellar granule cells
*Correspondence to: Giuseppe Legname; Email: legname@sissa.it
Submitted: 01/07/12; Revised: 03/12/12; Accepted: 03/13/12
http://dx.doi.org/10.4161/pri.20026
Introduction
Prion diseases are fatal neurodegenerative disorders that
include Creutzfeldt-Jakob disease, fatal familial insomnia and
Gerstmann-Sträussler-Scheinker disease in humans. Prions also
cause bovine spongiform encephalopathy in cattle, scrapie in
sheep and chronic wasting disease in deer, elk and moose. The
crucial event in these maladies is a post-translational transition,
in which the host-encoded cellular prion protein (PrP), denoted
PrPC, is transformed into a pathological conformer (PrPSc).1 PrPSc
is highly enriched with β-sheet structures.2
Encoded by Prnp gene, PrPC is a glycoprotein highly expressed
in the CNS. Although it is evolutionarily conserved among dif-
ferent classes of organisms, its function is still elusive. The
generation of PrP-null mice (Prnp0/0) failed to show any gross
phenotypes.3 Several PrP-knockout lines were produced and
some (Ngsk, Rcm0, ZrchII and Rikn) developed late-onset
ataxia accompanied by cerebellar neurodegeneration.4-6 Further
analysis demonstrated that the gene-knockout process employed
in the generation of such lines resulted in the ectopic expression
of a gene located 16 Kb downstream of Prnp locus.5 This gene,
later named Prnd, encodes a PrPC paralog protein called doppel
Doppel (Dpl) protein is a paralog of the prion protein (PrPc) that shares 25% sequence similarity with the c-terminus of PrPc,
common N-glycosylation sites and a c-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor.
Whereas PrPc is highly expressed in the central nervous system (cNs), Dpl is detected mostly in testes and its ectopic
expression in the cNs leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism
through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from
postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular
events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced
apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were
incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration.
similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates
apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated
apoptosis.
alessandro Didonna,1,† Joshua sussman,2,# Federico Benetti1,‡ and Giuseppe Legname1-3,*
1Department of Neuroscience; Laboratory of Prion Biology; scuola Internazionale superiore di studi avanzati (sIssa); Trieste, Italy; 2Institute for Neurodegenerative Diseases
and Department of Neurology; University of california; san Francisco, ca Usa; 3eLeTTRa Laboratory; sincrotrone Trieste s.c.p.a.; Trieste, Italy
†current address: Davee Department of Neurology; Northwestern University Feinberg school of Medicine; chicago, IL Usa; ‡european center for the sustainable Impact of
Nanotechnology; Rovigo, Italy; #cognition and Development; University of california; Berkeley, ca Usa
Keywords: prion, doppel, apoptosis, Bax, caspase, neurodegeneration
Abbreviations: PrP, prion protein; PrPC, cellular isoform of PrP; PrPSc, scrapie isoform of PrP; Dpl, doppel
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
(Dpl), most likely derived from the duplication of a single ances-
tral gene.7
Dpl resembles an N-terminally truncated form of PrP and
has been found to share several structural and biochemical fea-
tures with PrPC.8,9 Although Dpl primary sequence has only
25% homology with the C-terminus of PrPC, both proteins
are structurally similar as confirmed by NMR studies.10 Their
structures are characterized by three α-helices and two short
antiparallel β-strands.11,12 While PrPC structure is stabilized by
a single disulfide bond (Cys 178–Cys 213, mouse numbering),
Dpl tertiary structure presents two disulfide bonds: one at Cys
109–Cys 143 and the other between Cys 94 and Cys 148. The
higher stability conferred by the presence of an additional disul-
fide bond may explain why Dpl does not undergo the conforma-
tional change seen in PrP.13 Moreover, both proteins have two
N-glycosylation sites and a glycosylphosphatidyl inositol (GPI)
anchor that allows proper localization in lipid rafts. Despite
these biochemical similarities, the expression patterns of PrPC
and Dpl are extremely different. While PrPC is highly abun-
dant in the CNS, Dpl localizes mainly in non-nervous tissues,
especially in testes.14 In fact, Dpl seems to be involved in sper-
matogenesis and sperm-egg interaction since transgenic mice

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showed similar apoptotic features, fully rescued by full-length
PrP co-expression.23-25
The deletion of N-terminal residues 23–88 from PrP seems
to interfere with the rescue of ΔPrP/Dpl-induced neurodegen-
eration.26 Moreover, Prnp0/0 mice expressing a chimeric protein
composed of Dpl and the PrP N-terminus (1–124) failed to
exhibit any neurodegenerative signs.27 The same fusion pro-
tein, expressed in PrP-defective HpL3–4 cells, conferred resis-
tance against serum deprivation-induced apoptosis.28 Taken
together, these data suggest a crucial role for the octapeptide
repeat region and hydrophobic region for the anti-apoptotic
activity of PrP.
Although Dpl-induced apoptosis in the cerebellum has been
well characterized histopathologically, the molecular mecha-
nisms that occur in Purkinje and granular cells expressing Dpl
are still controversial. For this reason, we set up an in vitro system
to investigate molecular mechanisms of Dpl-induced cerebel-
lar neurodegeneration and dissect the pathways involved in the
process. Primary cell cultures of cerebellar neurons from both
wild-type (wt) FVB and FVB/Prnp0/0 mice were tested for cell
viability after incubation with mouse (Mo) Dpl (26–155), full-
length MoPrP(23–230), or truncated MoPrP(89–230). Dpl-
specific apoptosis in cerebellar neurons was confirmed, as well
as its rescue upon co-incubation with full-length PrP, but not
truncated PrP(89–230). The role of Bax in triggering apoptotic
signals, as well as the involvement of caspase-3 in Dpl-induced
apoptosis were also demonstrated.
2 Prion Volume 6 Issue 3
Results
Analysis and characterization of MoDpl and MoPrP proteins.
Monomeric MoDpl and MoPrP proteins were expressed in E. coli
using high-density culture fermentations. The proteins, local-
ized in the inclusion bodies, were purified and characterized as
described in the Material and Methods section. SDS-PAGE fol-
lowed by silver staining and mass spectrometry was performed
for estimating protein quality. All proteins revealed, from the
gel analysis, a single band at the expected molecular weight and
were folded predominantly in an α-helical conformation as deter-
mined by CD spectroscopy (data not shown).
Dose-dependent neurotoxic effect of MoDpl on cerebellar
granule neurons. To study Dpl-induced apoptosis and the rela-
tionship between PrP and Dpl, we elected to use in vitro primary
cell cultures of cerebellar granule cells. Cerebellar cultures have
extensively been used as a model to study apoptotic mechanisms29
as well as to test the effects of anti-prion molecules in primary
neuronal cultures.30 Cultures from both wt FVB and FVB/
Prnp0/0 mice (P6) were incubated for 3 d with increasing con-
centrations of MoDpl, up to 100 μg/mL (~6 μM). Cell survival,
tested by calcein AM dye assay, was inversely dependent on the
MoDpl dose in both cell lines (Table 1). This toxic effect was
most evident at the higher concentrations of the protein, 50 and
100 μg/mL (~3–6 μMDpl). Cells incubated for 3 d with similar
concentrations of MoPrP(23–230) showed no toxic effects.
The apoptotic effect of Dpl was confirmed using TUNEL
assay, according to manufacturer’s protocol (Fig. S1). Cerebellar
granule cells of both wt FVB and FVB/Prnp0/0 mice were incu-
bated for 2 d in either media alone, 4 μM MoDpl or 4 μM
MoPrP(23–230), stained for TUNEL and counterstained with
DAPI to visualize total chromatin. Green-labeled cells were
classified as apoptotic cells and blue cells were classified as
healthy cells. A third category of cells was classified as “inde-
terminate” as some cells were clearly neither labeled green nor
blue. Stained cells were assessed under a microscope, using a
counting 1 mm2 graticule, to ensure all cells were analyzed.
In both wt FVB and FVB/Prnp0/0 cell cultures incubated with
MoDpl, > 75% of cells were apoptotic and < 20% of cells were
classified as healthy. Upon incubation with MoPrP(23–230),
wt FVB and FVB/Prnp0/0 apoptotic cells ranged from 10% to
20%, comparable to those obtained incubating with media
alone. A certain amount of cell death in control conditions is
probably due to enzymatic and mechanical stresses induced
by the techniques used to dissociate neuronal cells. Moreover,
the slight differences between the two assays depend on the
fact that they look at different phenomena. While calcein AM
discriminates between cells with and without a functional cell
membrane which is still capable of actively transporting the dye
inside the cytosol, TUNEL detects DNA fragmentation in the
nuclei of cells undergoing apoptosis.
To assess whether Dpl-induced apoptosis was specific to gran-
ule cell neurons, primary cultures of hippocampal neurons from
both wt FVB and FVB/Prnp0/0 were incubated with 3 μM of
MoDpl(26–155), MoPrP(23–230) or MoPrP(89–230) for 5 d,
then assessed for cell survival (Fig. S2). None of the recombinant
lacking Prnd gene exhibited infertility due to impaired acroso-
mal function.15
Since Dpl was discovered, much interest has been shown in
Dpl research. In particular, the study of Dpl-induced cerebel-
lar neurodegeneration has elucidated the importance of the
N-terminal domain of PrP in neuroprotection. In fact, expression
of N-terminally truncated PrP [PrP(Δ32–121) or PrP(Δ32–134)]
in Prnp0/0 mice16 leads to ataxia and Purkinje cell loss in the same
fashion as Dpl ectopic expression.17 The cytotoxic effects of ΔPrP
and Dpl are counteracted by full-length PrP,18-20 suggesting com-
mon molecular mechanisms, most likely interfering in some cel-
lular pathways essential for cell survival in which full-length PrP
is involved.21,22 The same results were recapitulated in several
neuronal cell models. Human SH-SY5Y and murine neuroblas-
toma (N2a) cell lines transiently expressing Dpl or PrP(Δ32–121)
Table 1. MoDpl induces cell death in granule cells
MoDpl (µM)
wt FVB FVB/Prnp0/0
0101.7 ± 0.7 92.8 ± 3.8
0.3 76.7 ± 2.488.3 ± 4.7
3 39.0 ± 1.046.7 ± 1.0
6 22.6 ± 9.4 29.9 ± 4.3
Primary granule neurons from wt FVB and FVB/Prnp0/0 mice were incu-
bated with increasing concentrations of MoDpl (0, 0.3, 0.6 and 6 µM)
and assessed for cell viability by calcein aM assay. survival is expressed
as a percentage of viable cells relative to medium-treated controls. Data
represent mean and standard error, respectively, from at least three
independent measurements.

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ebellar neurons were exposed to both MoPrP(23–230) and
MoDpl(26–155), a significant increase in cell survival (Fig. 1A,
p < 0.01) was observed. Cells were incubated with Dpl alone
(3 μM or 9 μM) or with 3 μM PrP, then assessed for cell viability
with calcein AM. Cell survival with 3 μM and 9 μM of Dpl alone
was ~60 and ~50%, respectively, which was fully rescued after
co-incubation with 3 μM MoPrP(23–230). As a control, aniso-
mycin was used. Anisomycin is an inhibitor of DNA synthesis,
which is known to be toxic to cells in a PrPC-independent manner.
When MoPrP(89–230), which lacks the N-terminal sequence,
was co-incubated with MoDpl(26–155), no rescue was observed
(Fig. 1B). Thus, full-length MoPrP(23–230) appears to be critical
for the rescue of Dpl-induced toxicity of cerebellar neurons.
Dissecting the mechanism of Dpl-induced apoptosis. To
define and dissect the pathway involved in Dpl toxicity, we
studied the role of Bax in mediating the apoptotic process. We
prepared primary cultures from cerebellar granule neurons from
wt Bax and Bax-/- mice. Cultures were incubated for 5 d, with
MoDpl(26–155) and assessed for cell survival by calcein AM.
Cerebellar granule neurons from wt Bax mice revealed dose-
dependent cell death upon incubation with MoDpl(26–155)
(Fig. 2, shaded bars). In comparison, granule cells derived from
Bax-/- mice were unaffected by the same concentrations of MoDpl
(Fig. 2, open bars). Anisomycin, a potent inducer of apop-
tosis through the Bax pathway,34 was used as positive control.
Therefore, in the absence of the Bax gene, apoptosis induced by
either Dpl or anisomycin was prevented (Fig. 2).
www.landesbioscience.com Prion 3
Involvement of caspase-3 in Dpl-induced apoptosis. The
apoptotic process can follow two different pathways downstream
of Bax: caspase-dependent and caspase-independent route.
Caspase-3 has been identified as a key molecule in mediating
apoptosis in mammalian cells.35 A selective and irreversible phar-
macological inhibitor of caspase-3, AC-DEVD-CMK, was used
to assess its involvement in Dpl-induced apoptosis (Fig. 3).
For cerebellar cultures from wt FVB mice (Fig. 3A), cell sur-
vival in the presence of 9 μM of MoDpl was ~40%. With the
addition of the caspase-3-inhibitor, cell survival increased signifi-
cantly to 55% (p < 0.05). Similar observations were made for cer-
ebellar cultures derived from FVB/Prnp0/0 mice (Fig. 3B). As a
positive control, camptothecin was used. This inhibitor of DNA
synthesis is known to be toxic to cells. These results demonstrate
that inhibition of caspase-3 increased the survival of cerebellar
neurons, suggesting that caspase-3 plays a role in Dpl-induced
apoptosis.
proteins appeared to influence the survival of the hippocampal
neurons when compared with the media control.
Differential rescue by co-incubation with MoPrP(23–230).
In 2001, Moore et al.31 demonstrated in transgenic mice that full-
length PrP rescued Dpl-induced neurotoxicity in vivo. In order
to investigate the interaction between the two proteins, ELISA
experiments were performed. We found that MoDpl bound to
immobilized MoPrP in a direct ELISA (Fig. S3) as detected
using a rabbit polyclonal antibody against Dpl. Furthermore,
both MoPrP(23–230) and MoPrP(89–230) bound to immobi-
lized MoDpl, as detected by a rabbit polyclonal antibody to PrP
(Fig. S3). Interestingly, full-length MoPrP(23–230) showed a
greater binding capacity to Dpl compared with MoPrP(89–230),
which lacks the octapeptide region. The absence of the octare-
peat sequences in MoPrP(89–230) therefore could account for
the lower binding to MoDpl and may be influential in the ability
of PrP to rescue Dpl-induced apoptosis.
To validate the data obtained with recombinant proteins
expressed in bacteria, another source of MoPrP, a dimeric form
of PrP expressed in a eukaryotic system [murine neuroblastoma
(N2a) cells], MoPrP-Fc,32 was used. MoPrP-Fc protein bears all
the post-translational modifications occurring in PrP in vivo,
such as glycosylation. Interestingly, MoPrP-Fc bound to Dpl as
effectively as full-length, monomeric MoPrP(23–230), confirm-
ing our previous results (Fig. S3). In addition, these ELISA data
are supported by surface plasmon resonance (SPR) data as pub-
lished by Benvegnù et al.33
We also tested whether MoPrP(23–230) could rescue Dpl
toxicity in primary cell cultures of granule neurons. When cer-
Figure 1. Dpl-induced toxicity is rescued by full-length PrP. Primary
granule cell cultures were co-incubated with equimolar and sub-equi-
molar concentrations of MoDpl and full-length MoPrP (a) or truncated
MoPrP (B). after 5 d, cell survival was assessed by calcein aM. Full-
length MoPrP was able to fully rescue Dpl-induced apoptosis whereas
no rescue was detected using truncated MoPrP. as positive and nega-
tive controls, anisomycin and media alone were used, respectively. The
data are from at least three independent experiments (**p < 0.01).

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similarity in their tertiary structures. In order to dissect the Dpl-
activated pathways in the cerebellum, we set up an in vitro assay
using primary post-natal mouse cerebellar granule cells from wt
FVB and FVB/Prnp0/0 animals. We utilized an E. coli-expressed,
monomeric MoDpl(26–155) polypeptide refolded in α-helical
conformation and lacking post-translational modifications such
as glycosylation.
First, we showed that incubation of granule cells with MoDpl
(3–6 μM) induced a diffuse apoptosis that was completely res-
cued by co-incubation with full-length MoPrP(23–230) but
not with N-terminally truncated MoPrP(89–230), consistently
with data derived from transgenic mouse models.36 We probed
the specificity of Dpl activity on cerebellum by incubating hip-
pocampal primary neurons with MoDpl (3 μM). No neuro-
toxic effects were detected upon Dpl exposure. The differential
response between these two neuronal populations toward Dpl
had been analyzed previously to some extent in the pionieristic
work of Legname et al. 2002.32 The authors used a chimeric form
of both Dpl and PrP—fused to the Fc domain of an immuno-
globuline—to search for their physiological ligands within the
CNS. Immunohistochemical analyses highlighted that both
PrP-Fc and Dpl-Fc restrictly bind to granule cells in the cerebel-
lum. These data are compatible with the presence of a receptor
for Dpl and PrP on granule cell bodies, which is not expressed
in hippocampal neurons. This hypothesis is corroborated by the
fact that the concentrations at which Dpl induces apoptosis are
low, within the micromolar range. This evidence suggests that its
4 Prion Volume 6 Issue 3
molecular mechanism of neurotoxicity is not due to accumula-
tion of the protein in the extracellular compartment but, most
likely, to specific interactions with a ligand on the cell surface of
granule neurons. According to our ELISA data, Dpl binds to PrP
and this interaction has been recently confirmed either by SPR
or by in vivo experiments.33,38 Moreover, Dpl has been shown
to interact distinctively with a plasma metalloproteinase inhibi-
tor, the α-2-macroblobulin (α2M), in the granule cell layer of
the cerebellum.32,33 According to the proposed mechanism, in the
absence of PrP, Dpl could bind and sequester α2M and the with-
drawal of proteinase inhibitors may eventually lead to cerebellar
degeneration. The presence of PrP would prevent Dpl-α2M bind-
ing and the subsequent apoptotic processes.
Our data are in agreement with this proposed mechanism,
which also might explain why in our system, Dpl induces apop-
tosis both in wt and PrP-deficient cells. This finding is apparently
in contrast with previous data, which showed an effect on survival
only in cells from PrP-knockout mice.37 Indeed, when recombi-
nant Dpl is in excess, all endogenous PrP in wt cells is virtually
bound; in this state of PrP inactivity, remaining Dpl is available
to sequester α2M and promote neurotoxicity. Presumably, genetic
overexpression of Dpl in mouse cerebella with a wt background
for PrP could not be sufficient to bind all the endogenous PrP.
Hence, those mouse models did not show any pathological phe-
notype. Interestingly, N-terminally truncated PrP showed lower
affinity to Dpl compared with full-length PrP in SPR experi-
ments and also lacked the ability to rescue Dpl-induced apopto-
sis in our cultured-cell experiments. These observations support
the involvement of the N-terminal domain in neuroprotection as
indicated in previous genetic experiments,39 suggesting that the
N-terminus of PrP may exert its function by sequestering Dpl
and rendering it unavailable to interact with metalloproteinase
inhibitors.
Second, we investigated the role of Bax in Dpl-mediated neu-
rodegeneration. Bax is a pro-apoptotic member of the Bcl-2 family
that regulates cell death in many cell types. Bax can form homodi-
mers or heterodimers with Bcl-2 itself. Heterodimerization with
Bcl-2 no longer protects the cell from programmed cell death
and inevitably leads to apoptosis.40 Previous studies focusing on
Purkinje cells (PCs) showed controversial results. The deletion of
Bax in Ngsk/Prnp0/0 mice resulted in a partial rescue of PCs num-
ber, suggesting that the ectopic expression of Dpl induces both
Bax-dependent and Bax-independent pathways of cell death.41
The same authors showed that the pro-apoptotic effects of Dpl
on PCs can be partially counteracted by Bcl-2 overexpression.42
Recently, they have also described the upregulation of autopha-
gic markers as well as extensive accumulation of autophagosomes
in PCs of Ngsk mice, proposing that a progressive dysregulation
of autophagy could contribute to PCs loss by triggering apop-
totic cascades.43,44 In contrast, other authors failed to detect any
ameliorating effect on PCs derived from Bax inactivation in
Tg(Dpl) mice in which Dpl is ectopically expressed in the CNS
driven by the neural-specific enolase promoter.45 Interestingly,
also truncated PrP-induced neurodegeneration in mice express-
ing PrP(Δ32–134) seems to be mediated by Bax. In particu-
lar, a double-step mechanism takes place in which Bax-related
Figure 2. The role of Bax in Dpl-mediated toxicity. Primary granule cell
cultures from wt Bax (shaded bars) and Bax-/- (open bars) mice were in-
cubated with 3 or 6 μM MoDpl and tested for cell viability by the calcein
aM assay. cells derived from wt Bax mice showed a dose-dependent,
Dpl-induced apoptosis whereas cells from Bax-knockout mice failed to
show any cytotoxic features. anisomycin was used as positive control.
The data represent means from at least three independent experiments
(**p < 0.01).
Discussion
Although the pro-apoptotic function of Dpl has been well
assessed in Purkinje and granule cells of cerebellum, the
molecular basis of the process remains still controversial. The
understanding of Dpl mechanisms may help in elucidating the
neuroprotective role of PrP since the two molecules share a high

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rial pellet was resuspended in 25 mM TRIS-HCl, 5 mM EDTA
(pH 8.0) and processed twice in a Microfluidizer M-110EH
(Microfluidics Corp., USA). Inclusion bodies were collected
by centrifugation and solubilized in five volumes of 8 M Urea,
10 mM MOPS (pH 7.0) by agitation overnight at room tem-
perature (RT). The protein was purified by column chromatog-
raphy using carboxyl methyl sepharose (Amersham Bioscience)
followed by C4 reverse-phase media (Phenomenex).
Recombinant truncated MoPrP(89–230) was expressed in E.
coli host 27C7 from plasmid pNT3A as described in reference
47. Insoluble inclusion bodies that contained PrP were extracted,
solubilized and purified by various chromatographic procedures
as described by Mehlhorn and colleagues.49
For the production of MoDpl(26–155), a bacterial construct
for the expression of MoDpl(26–155) in the expression vector
pET11a (Novagen) was generated by standard working proce-
dures. After transformation into E. coli (DE3) cells, fermented
cultures were processed in a Microfluidizer as described for
MoPrP. The protein was then purified using a Mono S FPLC
column with the peak fractions lyophilized and stored until use.
MoPrP-Fc was constructed by cloning the MoPrP sequence
(23–230) between Ndel and Xbal sites of pSecTag plasmid
(Invitrogen) containing the human IgG1-Fc region. As a con-
trol, Fc expressing pSecTag plasmid was also constructed. A
FLAG (DYK DDD DK) epitope tag was cloned into the 3' end
of either MoPrP-Fc or Fc constructs using the Quick-change
PCR mutagenesis kit (Strategene, USA). Neuroblastoma (N2a)
www.landesbioscience.com Prion 5
cells were transiently or stably transfected with DNA con-
structs (5 or 10 μg) using the DOTAP DNA transfection kit
(Boehringer Mannheim, Europe). Stably transfected N2a cells
with either MoPrP-Fc or Fc constructs were maintained as previ-
ously described in reference 32, with the media supplemented
with zeocin at 200 μg/mL.
To check the transfection efficacy, cells were lysed in buffer
T, which contains 10 mM TRIS-HCl, pH 8.0; 0.5% deoxycho-
late; 0.5% Nonidet P-40; 150 mM NaCl. Cell lysates or cell-
conditioned media containing the fusion protein were resolved
by SDS-PAGE. Samples were blotted onto PVDF membranes
and blocked with 5% (w/v) non-fat milk protein in Tris-buffered
saline with 0.05% Tween-20 (TBST). The Fc portion of the pro-
tein was detected using anti-human Fc antibodies conjugated to
horseradish peroxidase (SIGMA) at various concentrations in
TBST. Blots were developed with the enhanced chemilumines-
cence (ECL) reagent (Amersham) for 1 min and exposed to ECL
hypermax film (Amersham).
Purification of the fusion proteins was performed as follows.
After transfection, cells were cultured for a minimum of 72 h
with the conditioned media containing the secreted fusion pro-
teins. The media was centrifuged at 3,500x g to remove cellular
debris and loaded onto a column of anti-FLAG M2 affinity gel
(SIGMA). Affinity gel with the immobilized protein was washed
with 20 column volumes of PBS. Bound protein was then eluted
with 5 column volumes of PBS containing 100 μg/mL FLAG
peptide.
pathways mediate only the early neurotoxic actions
and Bax-independent pathways are co-responsible
for the overall neurodegeneration induced by the
truncated form.46
Our data presented here indicate that the absence
of Bax abolishes Dpl-induced apoptosis in granule
cells occurring through the activation of caspase-3.
The possible discrepancies in the sensitivity of PCs
and granule cells toward Bax deletion are not surpris-
ing. Different death pathways might be activated in
these two neuronal cell populations upon exposure
to Dpl, reflecting the expression of cell type-specific
Dpl co-receptors at the level of cellular membrane.
The availability of an in vitro model for Dpl-induced
neurodegeneration will nevertheless assist in the iso-
lation of these putative binding partners.
Materials and Methods
Recombinant protein production and purification.
Generation and purification of all proteins used in
this study were as described previously, in detail in
reference 47 and 48. To increase expression levels of
recombinant, full-length MoPrP(23–230), an ala-
nine was added after the initiator methionine.
Recombinant full-length MoPrP(23–230) was
expressed from pET11a plasmid in E. coli BL21
(DE3) (Novagen, Madison, WI USA) in minimal
media containing 100 μg/mL ampicillin. The bacte-
Figure 3. The role of caspase-3 in Dpl-mediated apoptosis. Granule cell cultures
from wt FVB (a) and FVB/Prnp0/0 (B) mice were co-incubated for 5 d with 9 μM MoDpl
and 100 μM ac-DeVD-cMK (an inhibitor specific for caspase-3) diluted in DMsO. The
cells co-incubated with ac-DeVD-cMK showed a significant increase in cell survival
compared with cells incubated with MoDpl only, suggesting that caspase-3 may be
involved in mediating Dpl-induced apoptosis. camptothecin (10 μM) and DMsO
(8 μL) alone were used as positive and negative controls, respectively. The data repre-
sent means from at least three independent experiments. (*p < 0.05).

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again into ice-cold Hank’s solution. Under a stereomicroscope
(Nikon SMZ 1500), the structures of interest were dissected,
cleansed of meninges if necessary (to avoid glial contamina-
tion), minced and transferred to a sterile, 15-mL conical tube
(BD Biosciences). This was then centrifuged at 228 g in a
Beckmann GS-6 centrifuge for 5 min. The supernatant was
carefully aspirated, mixed thoroughly with a solution contain-
ing 20 units of papain (Worthington) and 0.005% DNase
(Worthington) and incubated at 37°C for 20–45 min, depend-
ing on the tissue volume. Tissues were then mechanically disso-
ciated using 5 mL and 1 mL pipette tips. Dissociated cells were
passed through a 40 μm cell strainer (BD Falcon) further spun
at 228 g and resuspended in 3 mL of Neurobasal-A media (P6)
containing B-27 supplement; fetal bovine serum (1%); gluta-
max-1 (2 mM); penicillin and streptomycin G (100 U/mL).
The proportion of viable cells was determined by staining with
trypan blue (Sigma), visualized with a hematocytometer under
a light microscope and plated in complete Neurobasal media
(±A) at 1 x 105 cells per 12 mm2 coverslip or per well of a 96 well
plate. All coverslips or wells had been coated with poly-d-lysine
at 250 μg/mL.
Incubation with recombinant proteins. Approximately 24
h after plating of cells, 0.1% Arabinose C was added to fresh
media to suppress non-neuronal proliferation. Recombinant
proteins were initially resuspended in dH2O and diluted to
5 mg/mL. The solution was then diluted to 1 mg/mL with 8
M guanidine, and left to stand at RT for 30 min before rapid
6 Prion Volume 6 Issue 3
dilution to 0.1 mg/mL with Tris buffer (pH 8.0), dialysed
against 20 mM sodium acetate buffer at 4°C, and finally passed
through a 0.22 μm filter. For each experiment, the proteins
were added exogenously to each coverslip or well, using media
as the vehicle, and incubated for up to 5 d depending upon the
experiment.
Cell survival assay. Cell survival was assessed using calcein
AM, according to manufacturer’s protocol (Invitrogen). In brief,
1 mg of calcein AM was dissolved in 1 mL of DMSO to make
a stock solution. This solution was then diluted to 1:50 in Ca2+/
Mg2+-free Dulbecco’s PBS (Invitrogen). After 24 h, cells that had
been plated at 105 in 100 μl of Neurobasal-A medium on black,
96-well plates (BD Bioscience) were incubated between 2–5 d
with the recombinant proteins at 37°C. Cells were washed three
times using Ca2+/Mg2+-free Dulbecco’s PBS before being incu-
bated 1:10 with 100 μl of calcein AM in DMSO per well for
30 min. Viability of the cells was then read on a fluorescence
microplate reader (Tecan, USA) using an excitation filter of 490
± 10 nm and an emission filter of 530 ± 15 nm.
TUNEL staining. Apoptosis was determined using the
terminal deoxynucleotidyltransferase nick end-labeling assay
(TUNEL). Two days after treatment with MoPrP and MoDpl,
cells were fixed in 4% paraformaldehyde for 30 min. Before
pre-incubation and permeabilization with a solution containing
0.1% sodium citrate (Sigma) and 0.1% triton-X-100 (Sigma) for
10 min, cells were washed in PBS. After permeabilization, cells
were further washed for 5 min before staining using the Roche
Kit. Permeabilized cells were incubated 1 h at 37°C with 45 μL
of stain per coverslip. An additional two washes in PBS were
performed before mounting on slides in Vectashield media con-
taining DAPI (Vector Laboratories). Cells were viewed at x20
magnification on a Leica DMRB fluorescence microscope with
filters specific for DAPI and FITC at 490 nm.
Quantification of apoptosis. Quantification was performed
manually with a counting graticule (Ted Pella). Blue-labeled
nuclei indicated “healthy cells;” green-labeled nuclei indicated
apoptotic cells. For nuclei labeled both blue and green, a third
classification (indeterminate) was included to denote that cells
were putatively undergoing apoptosis.
Protein-protein interaction assay. To study Dpl and PrP
interaction, an ELISA assay was performed. Wells were pre-incu-
bated at 4°C for 1 h with a saturating solution containing 0.25%
bovine serum albumin and 0.05% Tween-20 in Ca2+/Mg2+-free
Dulbecco’s PBS. For each well, a defined amount of protein was
diluted in 100 μL of 0.1 M sodium bicarbonate solution and
incubated overnight at 4°C. After nine washes with 1x TBST,
wells were blocked using the saturating solution for 1 h at RT.
All subsequent incubations for protein binding were performed
at RT.
In general, indicated amounts of PrP were diluted in the
saturated solution and incubated for 2 h. Nine repeated washes
between incubations were performed with 1x TBST. For PrP
detection, either a 2 μg/mL of humanized anti-PrP HuM-D18
antibody fragment (Fab),53 or a 1:1,000 dilution of a rabbit
polyclonal anti-PrP R073,54 was added and incubated for 1 h.
For Dpl detection, a 1:1,000 dilution of the rabbit polyclonal
Purity of the proteins. Purity of the recombinant proteins was
estimated by SDS-PAGE followed by silver staining50 and mass
spectrometry (data not shown). Structural conformation of puri-
fied protein was also analyzed using CD spectroscopy (data not
shown) as described previously in reference 48. Immunochemical
analysis of the fusion proteins was performed by SDS-PAGE fol-
lowed by western blotting using a panel of antibodies directed
against various sites of both the PrP-Fc fusion protein and Fc
domain. Recombinant MoDpl(26–155), MoPrP(23–230) and
MoPrP(89–230) were solubilized in either distilled water or
refolded in 20 mM sodium acetate buffer (pH 5.5) and stored at
4°C until needed.
Primary cell cultures. For preliminary experiments, which
were performed to develop and evaluate the assay in a murine
model, primary cell cultures were obtained from both cerebellar
granule cell layer and hippocampal layer of wt FVB and FVB/
Prnp0/0 mice51 at postnatal day 6 (P6). In addition to wt FVB and
FVB/Prnp0/0 mice, cultures were obtained from wtBax and Bax-/-
mice52 at P6. All experiments were performed in accordance with
European regulations [European Community Council Directive,
November 24, 1986 (86/609/EEC)], and approved by the local
authority veterinary service.
Cell cultures were prepared as follows. Working as quickly as
possible and under sterile conditions, the P6 mice were decapi-
tated and the heads placed immediately into a Petri dish con-
taining ice-cold Hank’s BSS, Ca2+/Mg2+-free, without Phenol
Red (Invitrogen) solution containing a high concentration of
penicillin/streptomycin to slow metabolism and decrease exter-
nal contamination. The whole brain was removed and placed

Page 7

© 2012 Landes Bioscience.
Do not distribute.
Shigematsu K, Sugimoto T, et al. Loss of cerebellar
Purkinje cells in aged mice homozygous for a disrupted
PrP gene. Nature 1996; 380:528-31; PMID:8606772;
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Moore RC, Lee IY, Silverman GL, Harrison PM,
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Rossi D, Cozzio A, Flechsig E, Klein MA, Rülicke T,
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ence.286.5441.914.
9. Silverman GL, Qin K, Moore RC, Yang Y, Mastrangelo
P, Tremblay P, et al. Doppel is an N-glycosylated, glyco-
sylphosphatidylinositol-anchored protein. Expression
in testis and ectopic production in the brains of
Prnp(0/0) mice predisposed to Purkinje cell loss. J Biol
Chem 2000; 275:26834-41; PMID:10842180.
10. Mo H, Moore RC, Cohen FE, Westaway D, Prusiner
SB, Wright PE, et al. Two different neurodegenerative
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bbrc.2004.05.115.
www.landesbioscience.com Prion 7
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors are indebted to Ms. Diane Latawiec for technical
assistance. The authors wish to thank Dr. Stanley B. Prusiner
for critically reviewing the manuscript, Ms. Hang Nguyen and
Erica Sarnataro for their excellent editorial support and Mr.
Patrick Culhane for fermentation and purification of recom-
binant proteins. This work was supported by grants to G.L.
from the FondazioneCompagnia di San Paolo and the Italian
Ministerodella Salute.
Supplemental Material
Supplemental material can be found at:
www.landesbioscience.com/journals/prion/article/20026
anti-Dpl antibody E6977,32 was added and incubated for 1 h.
The appropriate secondary antibody, goat anti-human Fab
(1:1,000 dilutions) conjugated to AP to detect the Fab fragment
antibody or an anti-rabbit IgG conjugated to AP, was further
incubated with the proteins for 1 h. PrP-Fc was detected using
a goat polyclonal anti-human Fc antibody conjugated to AP at
1:1,000.
Caspase inhibitor assay. AC-DEVD-CMK, a specific cas-
pase-3 inhibitor, was purchased from Calbiochem and dissolved
in DMSO to the desired concentration. It was added at the start
of the experiment and co-incubated with MoDpl(26–155) for up
to 5 d. Camptothecin was used as positive control.
Statistical analysis. For cell survival studies, means and stan-
dard deviations were calculated for each group of experiments.
The differences between the experiments were compared using
the unpaired Student’s t-test.
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