 |
INTRODUCTION |
Prion-associated pathologies are transmissible diseases that all
have fatal issues (1, 2). The elucidation of their etiology has
revealed a fully original transmission mechanism. It is now generally
admitted that the pathological infectious mechanism is due to a protein
present ubiquitously in the brain (referred to as
PrPc)1 that
becomes deleterious after its biophysical conversion into a
protease-resistant protein referred to as PrPres or
PrP-scrapie (3). This "bio-transformation" and subsequent pathologies absolutely require the presence of PrPc because
the absence of endogenous PrPc totally precludes the
PrPsc-mediated infectivity and neurotoxicity (4, 5).
Most of the works on prions have focused on these intriguing
disease-related features, particularly their conformation properties in vitro (6-8) and the species barrier allowing or limiting
the propagation of the various prion strains (for reviews see Refs. 1
and 2). The invalidation of the PrP gene (Prnp) led
to the conclusion that the Prnp
/
mice
exhibit normal development and unaltered behavioral phenotype (9,
10).
Several clues of a possible participation of PrPc in
programmed cell death came from the identification of a significant but restricted sequence homology of PrPc octapeptide repeats
with the anti-apoptotic oncogene Bcl-2 protein and its ability to bind
to Bcl-2 in double hybrid approach (11-12). A recent study (13) also
indicated that prion protein could rescue human neurons from
Bax-induced apoptosis. Alternatively, the possibility that a restricted
sequence corresponding to the 106-126 part of the molecule could
elicit a cellular toxic response has also been extensively documented
(14-16).
It is noteworthy that the 106-126 domain of the prion protein has no
genuine physiological or pathological existence as it has never been
described as a catabolite of PrPc that would have been
proteolytically generated upon normal or pathological conditions.
Therefore, the "toxic" potential of the 106-126
peptide could either be not relevant to any normal or altered situation or, alternatively, could reflect a phenotype that
would be also triggered by the whole parent prion protein. The latter
hypothesis would be in contradiction with the above studies, suggesting
an anti-apoptotic PrPc-related phenotype, but would be in
line with the work of Westaway et al. (17), showing that
transgenic mice over-expressing "normal" wild type PrPc
exhibit degeneration in central and peripheral nervous system as well
as in skeletal muscle. Interestingly, the 106-126 domain appears to be
targeted by a set of proteolytic activities called disintegrins,
ADAM10 and ADAM17, that cleave PrPc at the 110/111
peptide bond and thereby "inactivate" the putative toxic core of
the PrPc protein (18). To establish a pro- or
anti-apoptotic PrPc-mediated phenotype, we have examined
the possible toxicity of PrPc and its putative involvement
in the control of cell death. We show that in TSM1 cells,
over-expressed and endogenous PrPc all trigger
p53-dependent caspase 3 activation leading to a
pro-apoptotic phenotype.
 |
EXPERIMENTAL PROCEDURES |
Transfected TSM1 Cells and p53-inactive Cells--
Murine TSM1
neuronal cells (19) stably expressing mouse 3F4-tagged
MoPrPc (3F4MoPrPc) were obtained and cultured
as previously described (18). PrPc- or p53-antisense
cDNAs (referred to as ASPrP or ASp53, respectively) were obtained
after transfection of 1 µg of antisense pcDNA3 vector bearing
3F4MoPrPc or p53 with DAC30 (Eurogentec). Doubly
transfected 3F4MoPrPc/ASp53 TSM1 cells were obtained after
transfection of 3F4MoPrPc-expressing cells with ASp53
cDNA. p53-inactive cells were obtained from ATCC (NCI-H1299 clone)
and cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum.
Western Blot Analyses--
Cells were scraped and homogenized in
lysis buffer (50 mM Tris-HCl, pH7.5, 150 mM
NaCl, 5 mM EDTA containing 0.5% Triton X-100 and 0.5%
sodium deoxycholate). Equal amounts of protein (50 µg) determined by
the Bradford method (20) were separated on 12% SDS-PAGE gels and
analyzed for their p53, active caspase 3, Mdm2, phospho-p38, and
PrPc immunoreactivities by Western blot and hybridization
with anti-p53 (mouse monoclonal, Pab24, Santa Cruz Biotechnology),
anti-active caspase 3 (rabbit polyclonal R&D System), mouse monoclonal
anti-Mdm2, anti-phospho-p38 (Promega, Charbonnières-les Bains,
France) and anti-PrPc (SAF32, Ref. 21),
respectively. The secreted N-terminal fragment (N1) of PrPc
was recovered after incubation for 8 h at 37 °C/5%
CO2 in serum-free medium. Medium was then incubated
overnight with 2 µg/ml SAF32 monoclonal and protein A-Sepharose
(Zymed Laboratory Inc.) and analyzed using sheep anti-mouse
peroxydase-conjugated secondary antibody (dilution 1:10,000, Amersham
Biosciences) as described (18).
Flow Cytometry Analysis--
Cells were grown in 6-well plates
and incubated for 15 h at 37 °C in the presence or absence of
staurosporine (0.5 µM). Cells were rinsed with
phosphate-buffered saline, resuspended with 750 µl of buffer (20 mM Tris, 0.1% tri-natrium citrate, 0.05% Triton X-100) containing 50 µg/ml propidium iodide (PI) and incubated overnight at 4 °C. The PI fluorescence of individual nuclei was measured using a FACSCalibur flow cytometer (CellQuest software; BD
Biosciences). Nuclei were characterized for their side angle scatter
and forward-angle scatter parameters. Red fluorescence due to PI
staining of DNA was then counted on a logarithmic scale. All
measurements were performed under identical conditions. This technique
allows discrimination of populations of fragmented nuclei from debris
and non-viable cells and also from intact diploid nuclei those show
higher fluorescence staining. The number of apoptotic nuclei is
expressed as a percentage of the hundred thousand events gated.
XTT Assay of Cell Viability--
Cells were grown in a
5% CO2 atmosphere in 96-well plates in a final volume of a
100 µl per well and incubated for 24 h at 37 °C in the
presence or absence of 2 µM of staurosporine. Briefly, XTT is metabolized by mitochondrial dehydrogenase to a water soluble formazan salt only by metabolically active viable cells. XTT
metabolizing activity was determined as previously described (22).
Caspase 3-like Activity Assay--
Cells were cultured in 6-well
plates for 15 h at 37 °C in the absence or presence of
etoposide, ceramide C2, or staurosporine (0.5 µM) or 15 µM of the Fyn kinase inhibitor PP2. In some cases, cells
were pre-incubated for 24 h with Ac-DEVD-al (100 µM)
or for 2 h with the p38 MAPK inhibitor SB203580 (1 µM, Calbiochem). Cells were then rinsed, gently scraped,
pelleted by centrifugation, then resuspended in 40 µl of lysis buffer
and analyzed as previously detailed (22).
p53 Transcriptional Activity--
The PG13-luciferase p53 gene
reporter construct (provided by Dr. B. Vogelstein) has been previously
described (23). One µg of PG13-luciferase was co-transfected with 1 µg of a
-galactosidase transfection vector (to normalize
transfections efficiencies) in TSM1 neurons and in PrP+/+
and PrP
/
primary cultured neurons. Forty-eight hours
after transfection, luciferase and
-galactosidase activities were
measured according to previously described procedures (23).
Statistical Analysis--
Statistical analyses were performed
with Prism software (Graphpad Software, San Diego, CA) using
Newman-Keuls multiple comparison test for one-way analysis of variance
and the unpaired Student's t test for pair-wise comparisons.
 |
RESULTS |
PrPc Over-expression Increases Basal and
Staurosporine-induced Toxicity and DNA Fragmentation in TSM1
Neurons--
We have obtained stable TSM1 transfectants
over-expressing 3F4MoPrPc (Fig.
1A, upper panel).
As expected, these cells secrete higher amounts of a 11-12-kDa
fragment (Fig. 1A, lower panel), the
immunological characterization of which previously led to its
identification as the N-terminal product (called N1) derived from
proteolysis of PrPc at the 110/111-112 peptide bond (18).
The over-expression of PrPc in TSM1 neurons increases both
basal and staurosporine-induced toxicity (Fig. 1B) and DNA
fragmentation (Fig. 1C) as measured by XTT and PI
incorporation, respectively. This phenotype has been observed for all
clones examined (not shown).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 1.
Caspase activation, enhanced toxicity, and
DNA fragmentation in PrPc-expressing TSM1 neurons.
A, over-expression of PrPc and its secreted
product N1 in 3F4MoPrPc-TSM1 neurons was measured by
Western blot with SAF32 as described under "Experimental
Procedures" (#1-3 represent three independent
clones). B, TSM1 viability in basal and
staurosporine-treated conditions. Mock-(black bars) or
PrPc-expressing (gray bars) cell lines were
treated without ( ) or with (+) staurosporine (Sts) then
assayed for their XTT metabolizing activity as described under
"Experimental Procedures." Activities are expressed as the
percentage of the control (mock cells in the absence of staurosporine).
Bars are the means ± S.E. of 9 independent experiments
(triplicate determinations). C, basal and
staurosporine-stimulated PI incorporation measured in the indicated
cell lines by fluorescence-activated cell sorter analysis (similar data
were obtained in 3 independent experiments). The number of apoptotic
nuclei is estimated as described under "Experimental Procedures."
D, caspase 3 activity and immunoreactivity (for details see
"Experimental Procedures") in basal ( ) and Sts (+) conditions in
mock-transfected (black bars) or PrPc-expressing
TSM1 neurons (gray bars). Bars represent the
Ac-DEVD-al-sensitive caspase 3-like activity and are the means ± S.E. of 7 determinations (duplicate determinations; ns,
non-statistically significant). E, effect of the indicated
apoptotic effectors on caspase 3 activity in mock-transfected
(black bars) or PrPc-expressing TSM1 neurons
(gray bars) measured as in D. Bars
represent the means ± S.E. of 4-7 (duplicate determinations).
F, caspase 3 activity (for details see "Experimental
Procedures") in basal ( ) and Sts (+) conditions in mock-transfected
(black bars) or PrPc-expressing TSM1 neurons
(grey bars) after PP2 pretreatment (for details see
"Experimental Procedures").
|
|
Staurosporine-induced Caspase 3 Activation is Increased by
PrPc Over-expression in TSM1
Neurons--
Over-expression of PrPc increases
staurosporine-stimulated but not basal Ac-DEVD-al-sensitive
Ac-DEVD-7AMC-hydrolyzing caspase 3-like activity in TSM1 neurons (Fig.
1D). This was accompanied by an augmentation of
staurosporine-induced active caspase 3-like immunoreactivity (Fig.
1D). It should be emphasized that PrPc
expression potentiated the increase of caspase 3 activity triggered by
several other apoptotic stimuli such as ceramide C2 and etoposide in
TSM1 (Fig. 1E). PP2, a selective inhibitor of kinases
belonging to the Src family, such as Lck and Fyn, (24) did not affect the extent of PrPc-induced staurosporine-stimulated caspase
3 activation in TSM1 neurons (Fig. 1F).
PrPc Antisense Approach Lowers Staurosporine-induced
Caspase 3 Activation and DNA Fragmentation in TSM1 Neurons--
To
examine the endogenous contribution of PrPc to
TSM1 susceptibility to staurosporine, we set up stable
antisense-PrPc (ASPrPc) TSM1 transfectants.
This antisense approach led to a drastic reduction of endogenous
PrPc expression and to a barely detectable N1 secretion
(Fig. 2A). Interestingly, the
staurosporine-stimulated, but not the basal caspase 3 activity observed
in mock-transfected TSM1 neurons, was drastically reduced in
ASPrPc-TSM1 cells (Fig. 2B).
Concomitantly, PrPc-antisense abolished the
staurosporine-induced augmentation of active caspase 3-like
immuno-reactivity (Fig. 2B). In line with these
observations, the staurosporine-induced DNA fragmentation was reduced
by about 50% in ASPrPc-expressing TSM1 neurons (Fig.
2C), a percentage that matched well the extent of the
reduction of caspase 3 activity (see Fig. 2B).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 2.
PrP-antisense lowers TSM1 responsiveness to
staurosporine. Antisense-PrPc-expressing TSM1 cells
(ASPrP) were obtained and cultured as described under
"Experimental Procedures." A, ASPrP cells express less
endogenous PrPc- and N1-like immuno-reactivities (3 independent clones are shown). B, caspase 3 activity and
immuno-reactivity (Casp.3; for details see "Experimental
Procedures") in basal ( ) and Sts (+) conditions in
mock-transfected (black bars) or ASPrP-expressing TSM1
neurons (empty bars). Bars represent the
Ac-DEVD-al-sensitive caspase 3-like activity and are the means ± S.E. of 7 experiments (duplicate determinations; ns, non-significant).
C, basal and staurosporine-stimulated PI incorporation
measured in the indicated cell lines by fluorescence-activated cell
sorter analysis. The procedure and estimation of the number of
apoptotic nuclei are described under "Experimental
Procedures."
|
|
PrPc-induced Caspase 3 Activation is
p53-dependent in TSM1 Neurons--
We examined the
putative contribution of the tumor suppressor oncogene p53 to the
PrPc-induced caspase 3 activation and toxicity by antisense
and pharmacological approaches. We set up TSM1 stable transfectants
expressing p53 antisense cDNA either alone (ASp53) or in
combination with 3F4MoPrPc
(3F4MoPrPc/ASp53)(see Fig. 4B). Our data show
that all 3F4MoPrPc-related phenotypes can be prevented by
antisense down-regulation of p53. Thus, Fig.
3A clearly shows that ASp53
drastically protects TSM1 cells from both basal and
staurosporine-stimulated PrPc-mediated toxicity measured by
the XTT assay (compare 3F4MoPrPc with
3F4MoPrPc/ASp53). Furthermore, ASp53 fully prevents the
3F4MoPrPc-induced DNA fragmentation (Fig. 3D,
compare 3F4MoPrPc-sts with
3F4MoPrPc/ASp53-sts). Finally, ASp53
totally reverses the staurosporine-induced caspase activation triggered
by 3F4MoPrPc (Fig. 3B, compare
3F4MoPrPc and
3F4MoPrPc/ASp53 transfectants).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 3.
PrPc-induced caspase 3 is p53-dependent. A, mock-,
3F4MoPrPc, or 3F4MoPrPc-TSM1 cells stably
transfected with p53 antisense cDNA (3F4MoPrP/ASp53), respectively,
were checked for their cell viability in the absence ( ) or presence
(+) of staurosporine (Sts) as described in the legend to
Fig. 1. B-D, TSM1 cells stably transfected with p53
antisense cDNA (ASp53 and 3F4MoPrP/ASp53, respectively) were
measured for their caspase activity (B and C) and
DNA fragmentation (D) in the absence ( ) or presence (+) of
staurosporine (B and D) or pifithrin-
(Pft, 10 µM, B). Bars
represent the means ± S.E. of 3-6 independent determinations
(carried out in duplicates).
|
|
We took advantage of the recent design of a specific p53 inhibitor,
pifithrin-
(25), to further confirm the role of endogenous p53 in
PrPc-mediated caspase activation. This pharmacological
approach allowed us to show that pifithrin-
abolishes the
3F4MoPrPc-mediated staurosporine-stimulated increase of
caspase 3 activity (Fig. 3C, compare
3F4MoPrPc and Mock
/+pft). Interestingly, pifithrin-
appeared inactive in
ASp53-TSM1 cells (Fig. 3C, compare ASp53
/+pft), indicating that the antisense and pharmacological
approaches indeed inhibited the same expected molecular target,
i.e. p53. This conclusion was reinforced by the fact
that pifithrin-
did not significantly modify caspase 3 activity
displayed by doubly transfected 3F4MoPrPc/ASp53-TSM1
neurons that remain at the level of pifithrin-
-treated mock- and
3F4MoPrP-transfected cells (Fig. 3C).
PrPc-expression Increases p53-like Immunoreactivity and
Activity--
Because PrPc-proapoptoptic phenotype
appeared p53-dependent, we examined whether
PrPc could also affect p53 expression and transcriptional
activity. First, our data show that TSM1 over-expressing
3F4MoPrPc displays higher endogenous p53-like
immunoreactivity than mock-transfected cells (Fig.
4A). This was also observed in
transfected HEK293 cells (Fig. 4E). Conversely,
ASPrPc-TSM1 cells exhibit lower p53-like immunoreactivity
(Fig. 4A). To correlate the modulation of p53
immunoreactivity with its biological activity, we examined
the p53 transcriptional activity by means of the
PG13-luciferase construct classically used as the p53 gene reporter
(23). Interestingly, p53 transcriptional activity was drastically
enhanced in 3F4MoPrPc-TSM1 cells (Fig. 4C) and
lowered in ASPrPc-expressing neurons (Fig. 4C).
As expected, ASp53-expressing cells exhibit lower p53 immunoreactivity
(Fig. 4B, compare ASp53 and Mock) and
totally prevents the p53 increase observed with singly transfected
3F4MoPrPc cells (Fig. 4B, compare
3F4MoPrP and 3F4MoPrP/ASp53 with
ASp53).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 4.
PrPc modulates p53-like
immuno-reactivity and transcriptional activity. p53-like
immuno-reactivity (A, B) and transcriptional
activity (C) were monitored as described under
"Experimental Procedures" in the indicated transfected TSM1
(A-C). Bars are the means ± S.E. of 4-6
independent experiments (duplicate and triplicate
determinations).
|
|
PrPc Controls p53 at a Post-transcriptional
Level--
We took advantage of a cell system in which p53 is
functionally deficient (26) to examine the ability of PrPc
to increase p53 expression in co-transfection experiments. Any PrPc-dependent increase in p53 could not be
explained by a transcriptional activation as the p53 cDNA is driven
by a cytomegalovirus promoter and therefore would imply
post-transcriptional PrPc-dependent p53
modulation. As expected, mock-transfected cells do not display
any p53 transcriptional activity (Fig.
5A). PrPc cDNA
transfection does not modify this null phenotype. However, we establish
that transient co-transfection of PrPc and p53 cDNAs
drastically potentiates the p53 transcriptional activity associated
with p53 cDNA transfection alone (compare p53 and
3F4/p53 in Fig. 5A). These data led us
to search for post-transcriptional modifications triggered by
PrPc that would have altered p53 transcriptional activity.
Mdm2 was recently shown to decrease p53 metabolic stability thereby
lowering its activity (27). We demonstrate here that Mdm2 fully
reverses the PrPc-induced increase of p53 transcriptional
activity (Fig. 5E).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
Post-transcriptional regulation of p53 by
PrPc. A, p53 transcriptional activity was
monitored by means of the PG13-luciferase reporter gene (see
"Experimental Procedures") in p53-deficient fibroblasts following
transient transfections with 3F4MoPrP (3F4) or wild type
p53 (P53) cDNA alone or combined
(P53/3F4). Bars are the means of
6 ± S.E. In E, Mdm2 cDNA alone (Mdm2)
or in combination with p53 (Mdm2/P53) or 3F4 and
p53 (3F4/Mdm2/P53) were analyzed for
p53 transcriptional activity as in A. Bars are
the means of 3 ± S.E. B and C,
phosphorylated-p38 MAPK immuno-reactivity was examined in mock and
3F4MoPrPc stably transfected TSM1 neurons (B)
and in p53-deficient fibroblasts following 3F4MoPrP (PrP)
and/or wild type p53 (P53) transient transfections
(C). D, mock or 3F4MoPrPc cells were
treated without ( ) or with (+) staurosporine (Sts) in
absence ( ) or presence of SB203580 (SB) then analyzed for
caspase 3 activity as described under "Experimental Procedures."
Bars are the means ± S.E. of 4 independent
experiments.
|
|
As p38 MAPK was reported to up-regulate p53 activity via upstream
decrease of Mdm2 expression (28), we examined the effect of a selective
p38 MAPK inhibitor (29) and the endogenous levels of the active
phosphorylated counterpart of p38 MAPK. SB203580 significantly reduced
but did not abolish the staurosporine-induced caspase activation in
mock- and 3F4MoPrPc-expressing neurons (Fig.
5D). Accordingly, over-expression of PrPc
increases phospho-p38 MAPK immunoreactivity in both TSM1 neurons (Fig.
5B) and p53-inactive fibroblasts (Fig. 5C).
Altogether, our data suggest that PrPc increases p53
activity by p38 MAPK activation and concomitant reduction of Mdm2.
 |
DISCUSSION |
Although a series of studies (1-9) attempted to delineate
the prions-related pathogenic mechanisms, very few data concern the
putative physiological function of PrPc. This lack of
attention perhaps came in part from the fact that the absence of the
protein does not seem absolutely crucial because mice in which
the prnp gene had been invalidated are safe and healthy, with apparently poorly detectable alterations in their development and behavior (9). However, Kuwahara et al. (30) reported that hippocampal neurons prepared from
Prnp
/
mice undergo exacerbated cell death
triggered by serum deprivation, thereby suggesting that
PrPc could display an anti-apoptotic tonus. More recently,
an interesting study documented the fact that PrPc
protected human neurons from Bax-induced cell death (13). These data
are apparently in opposition with a previous work demonstrating that
transgenic mice over-expressing wild type PrPc could
exhibit severe neurodegeneration (17). On the other hand, a recent
article indicated that the post-natal deletion of the PrPc
alters CA1 hippocampal neuron excitability but does not induce neurodegeneration (10) as would have been expected if the protein displayed an important anti-apoptotic function at adulthood.
Furthermore, an infected hypothalamic neuronal cell line exhibits
increased apoptosis (31). Whether this results from a dysfunction in
the normal control of cell death by PrPc remains a possibility.
The 106-126 domain of PrPc was shown to be toxic (14, 15)
and appears to activate several pro-apoptotic markers (16, 32). This
fragment is never proteolytically released as such from
PrPc. In this context, either the bulk of studies examining
the effect of 106-126 just describe not relevant and artifactual data,
or, more likely, the studies carried out with this fragment can be seen
as a revelator of a PrPc function that would be
pro-apoptotic. Our study clearly establishes by means of neuronal cell
systems, stable transfections, antisense, and pharmacological
approaches that PrPc drastically exacerbates cell
responsiveness to pro-apoptotic stimuli. Thus, over-expression or
specific induction of PrPc enhances staurosporine-induced
cell toxicity, DNA fragmentation, and increases both caspase 3-like
activity and immuno-reactivity that are all reversed by
PrPc antisense approach. The fact that
PrPc-mediated caspase activation also occurs in HEK293
cells (33) importantly indicates that the pro-apoptotic
PrPc-related phenotype is not cell-specific.
A recent study (34) suggested that PrPc could behave as a
signaling molecule, transducing the signal after its coupling to the
Src kinase Fyn. In our experiments, the selective Src inhibitor PP2
does not affect the PrPc-induced caspase activation,
indicating that Fyn kinase phosphorylating activity was clearly not
essential for PrPc-induced apoptosis in our experimental
conditions. It should be noted that distinct cell types were used in
the two studies, and more particularly the Fyn-dependent
PrPc-mediated signaling appeared drastically dependent on
the differentiation state of the 1C11 cells used in Mouillet-Richard
et al. (35). Clearly the lack of Fyn dependence
observed for PrPc-mediated phenotype in TSM1 cells
indicates that Fyn requirement is likely restricted to few specialized
PrPc phenotypes much more than for more general PrP
function in cell death control.
We demonstrate that the PrPc-induced caspase 3 activation
is mediated by p53. Indeed, PrPc expression increases p53
immuno-reactivity and transcriptional activity, whereas the
PrPc antisense approach led to the opposite phenotype.
Furthermore, p53 antisense blocked the PrPc-induced
toxicity, DNA fragmentation, and caspase 3 activation. The opposite
phenotype triggered by down-regulation of endogenous PrPc
importantly indicates that the pro-apoptotic phenotype observed in
PrPc-transfected cells was not artifactually related to the
procedure involving over-expression of the protein. This is in
agreement with our other data showing that Rov9 cells display a
pro-apoptotic phenotype only after selective PrPc induction
(33).
PrPc controls p53 expression and activity at a
post-transcriptional level. Thus we were able to demonstrate that in a
cell line deficient in active p53, PrPc drastically
increases p53 activity after co-transfection of p53 and
PrPc cDNAs. As p53 and PrPc cDNA are
driven by a constitutively active cytomegalovirus promoter, p53
enhanced activity cannot be derived from increased transcription of
p53, the genuine promoter of which leads to an inactive protein in our
cell system. Interestingly, Mdm2 fully prevents the
PrPc-induced p53 increase observed in the above cell
system. Mdm2 was recently shown to control p53 expression at
post-transcriptional levels (27) and particularly modulates p53
ubiquitination and degradation. Therefore, one could conclude that
PrPc increases p53 expression and transcriptional activity
via a decrease of the p53 down-regulator Mdm2. To support this
hypothesis we examined the putative contribution of the p38
mitogen-activated protein kinase that was recently shown to
down-regulate Mdm2 and concomitantly increase p53 expression in neurons
in a model of hypoxia. Indeed, we clearly showed that phospho-38 MAPK
immuno-reactivity, the activated form of this kinase, was higher in
PrPc-expressing TSM1 cells, in PrP+/+ than in
PrP
/
neurons, and could be increased by
PrPc in fibroblasts (see Fig. 5). Furthermore, p38
inhibitor could partly block the PrPc-associated caspase 3 activation in transfected TSM1 neurons. Altogether, our data show that
PrPc exerts a pro-apoptotic phenotype through the
modulation of caspase 3 activity via a p53-dependent
pathway that is positively and negatively controlled by p38 MAPK and
Mdm2, respectively.
It is interesting to note that a recent study indicated that misfolded
PrPc could be neurotoxic and could induce
neurodegenerescence when accumulating in the cytosol, even at small
amounts (35). Whether such a small amount of PrPc could
account for the phenotype observed in our over-expression system is
difficult to examine. However, the fact that our antisense approach led
to an anti-apoptotic phenotype would suggest that even in normal
conditions, a small fraction of endogenous PrPc could be
responsible for our observed p53-dependent caspase 3 activation.
We recently documented the fact that PrPc
undergoes basal and protein kinase C-regulated cleavage in the middle
of its 106-126 domain by proteases of the disintegrins family, thereby
releasing a fragment that we called N1 (for review see Ref. 36). This PrPc cleavage also observed by Chen et al.
(37) is reminiscent of the one taking place in the middle of the
A
domain borne by the
-amyloid precursor protein (38).
Interestingly, this intra-A
cleavage not only lowers A
production
but also gives rise to a cytotrophic and neuroprotective secreted
fragment called sAPP
. Whether the cleavage generating N1 not only
abolishes a pro-apoptotic control of PrP but also produces a
fragment with opposite neuroprotective influence is currently being
examined in our laboratory.