Induction of neuronal apoptosis by expression of Hes6 via p53-dependent pathway.

Abstract

Hes6 is a member of hairy/enhancer of split (Hes) family that plays a role in the cell proliferation and differentiation. Recently, we found that Hes6 is involved in the regulation of cell proliferation via p53-dependent pathway. In addition to the proliferating regions, brain regions where early post-mitotic neurons are enriched also exhibited Hes6 and p53 mRNA expression. Because p53 is involved in the post-mitotic neuronal apoptosis, here we investigated whether Hes6 can influence the neuronal survival/death. Overexpression of wild-type Hes6 and its mutants induced the apoptosis of primary cultured cortical neurons. In addition, neuronal apoptosis by Hes6 overexpression was markedly blunted in p53(-/-) or Bax(-/-) cortical neurons, suggesting that these pro-apoptotic effects are mediated by p53- and Bax-dependent pathway. However, transactivation-defective mutants of Hes6 also enhanced neuronal apoptosis, suggesting that apoptogenic activity of Hes6 is not directly related to its role in the transcriptional regulation. We propose that Hes6 may play a significant role in the neuronal cell death and/or pathological neurodegeneration via activation of p53 signaling.

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Research Report
Induction of neuronal apoptosis by expression of Hes6 via
p53-dependent pathway
Bokkee Euna, Bongki Choa, Younghye Moonb, Soo Young Kima, Kyungjin Kimb,
Hyun Kima,⁎, Woong Suna,⁎
aDepartment of Anatomy, Brain Korea 21, Korea University College of Medicine, Seoul, Korea
bSchool of Biological Science, Seoul National University, Seoul, Korea
A R T I C L E I N F OA B S T R A C T
Article history:
Accepted 30 November 2009
Available online 5 December 2009
Hes6 is a member of hairy/enhancer of split (Hes) family that plays a role in the cell
proliferation and differentiation. Recently, we found that Hes6 is involved in the
regulation of cell proliferation via p53-dependent pathway. In addition to the
proliferating regions, brain regions where early post-mitotic neurons are enriched also
exhibited Hes6 and p53 mRNA expression. Because p53 is involved in the post-mitotic
neuronal apoptosis, here we investigated whether Hes6 can influence the neuronal
survival/death. Overexpression of wild-type Hes6 and its mutants induced the apoptosis
of primary cultured cortical neurons. In addition, neuronal apoptosis by Hes6
overexpression was markedly blunted in p53−/−or Bax−/−cortical neurons, suggesting
that these pro-apoptotic effects are mediated by p53- and Bax-dependent pathway.
However, transactivation-defective mutants of Hes6 also enhanced neuronal apoptosis,
suggesting that apoptogenic activity of Hes6 is not directly related to its role in the
transcriptional regulation. We propose that Hes6 may play a significant role in the
neuronal cell death and/or pathological neurodegeneration via activation of p53 signaling.
© 2009 Elsevier B.V. All rights reserved.
Keywords:
Hes6
Neuronal cell death
Nuclear
Mitochondria
1.Introduction
Basic-helix–loop–helix (bHLH) proteins are a superfamily of
DNA-binding transcription factors that regulate numerous
biological processes such as determination and differentiation
of specific cellular lineages (Guillemot, 1999; Kageyama et al.,
2005). Among these, hairy/enhancer of split (Hes) family of
bHLH proteins acts downstream to Notch signaling which
suppresses cell differentiation (Fischer and Gessler, 2007;
Kageyama et al., 2005). For instance, Hes1 and Hes5 are
expressed in the ventricular zone of the telencephalon where
they maintain progenitors in an undifferentiated, proliferative
state and inhibit the differentiation of progenitors into
neurons (Ohtsuka et al., 1999; Sasai et al., 1992). On the other
hand, another Hes family member, Hes6 conversely promotes
neuronal differentiation and inhibits differentiation into
astrocytes in vitro (Cossins et al., 2002; Jhas et al., 2006). This
function of Hes6 is known to be mediated by the suppression
of Hes1 by the interaction with corepressor Groucho/TLE or by
the promotion of proteolytic degradation of Hes1 (Gratton et
al., 2003). Recently, we found that Hes6 is a component of
promyelocytic leukemia (PML)-nuclear body (NB) complex,
B R A I N R E S E A R C H 1 3 1 3 ( 2 0 1 0 ) 1 – 8
⁎ Corresponding authors. Fax: +82 2 929 5696.
E-mail addresses: kimhyun@korea.ac.kr (H. Kim), woongsun@korea.ac.kr (W. Sun).
Abbreviations: Hes6, Hairy/enhancer of split 6; VZ, ventricular zone; PVR, paraventricular region; EGL, external germinal layer; Cbll,
cerebellum; Hip, hippocampus; NLS, Nuclear localization signal; CBP, CREB binding protein; (PML), Promyelocytic leukemia; HD,
Huntington's disease
0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2009.11.078
available at www.sciencedirect.com
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and it can arrest cell cycle via promoting p53 activity (Eun et
al., 2008), which is required for cell differentiation. Hes6
associates with PML-NB via its C-terminal WRPW domain,
anddirectlybindsto anotherPML-NBprotein,CBP,via its basic
domain. Association of Hes6 with CBP promotes the acetyla-
tions of p53 and histones surrounding p21 promoter region,
which in turn enhances the expression of p21 cell cycle
inhibitor gene (Eun et al., 2008).
Inneurons, p53 isalso involvedinthe regulation of neuronal
apoptosis as a transcription factor that promotes the transcrip-
tion of genes triggering apoptotic cascade (e.g., Bax, PUMA and
Noxa) (Cregan et al., 1999; Culmsee and Mattson, 2005; Jacobs et
al., 2006; Jeffers et al., 2003). At early stage of neuronal
differentiation, p53 accumulates in the nucleus, where it
activates genes involved in cell differentiation. However, once
cells become committed to become neuron, p53 no longer
maintains in the nucleus and localizes in the cytoplasm and
otherorganellessuch as themitochondria(Milleret al.,2000).In
themitochondria,ithasbeenreportedthatp53canpromotecell
death by the direct promotion of pro-apoptotic gene Bax
activation (Miller et al., 2000). These nuclear and cytoplasmic
p53-mediated neuronal deaths are critical events in the
neurodegenerative diseases, including Alzheimer's disease
(AD), Parkinson's disease (PD) and Huntington's disease (HD)
(Bae et al., 2005; Feng et al., 2006; Lin and Beal, 2006). For
instance, p53 regulates the transcription of huntingtin (Feng et
al., 2006). In addition, retinal degeneration by the expression of
mutant huntingtin (mHtt) is diminished in p53-null mutant
flies, and behavioral abnormalities by mHtt are abolished in
p53-knockout mice (Bae et al., 2005).
Considering that Hes6 plays roles in the regulation of p53
function, we hypothesize that Hes6 may be involved in the
control of post-mitotic neuronal apoptosis. Here, we identified
that both Hes6 and p53 are expressed in early post-mitotic
neurons of embryonic and adult brain, and overexpression of
Hes6 promotes p53-dependent neuronal death in vitro. These
results support the idea that Hes6 is involved in the p53-
dependent neuronal deathunder physiological or pathological
conditions.
2.Results
2.1.
post-mitotic region in developing rat brain
Expression of Hes6 and p53 in proliferating and early
The expression pattern of Hes6 mRNA in developing and adult
brain was explored by in situ hybridization histochemistry
(Fig. 1). On embryonic day 16 (E16), Hes6 mRNA was mainly
distributed in the ventricular zone containing proliferating
neural precursor cells. On early postnatal stages (P0–P7), a
moderate level of Hes6 signal was maintained in the
proliferating regions such as paraventricular region (PVR),
and external germinal layer (EGL) of cerebellum (Cbll). Hes6
mRNA was also observed in brain regions where post-mitotic
neurons enriched, such as the hippocampus (Hip) and the
upper layer of the cerebral cortex. With decrease in the
proliferating/early post-mitotic neuronal population, Hes6
expression disappeared in most adult brain regions. However,
a weak but substantial expression of Hes6 was monitored in
the dentate gyrus (DG) where adult neurogenesis persists in
adulthood (Jhas et al., 2006; Lledo et al., 2006). These results
indicate that Hes6 is expressed in neural precursor cells and
their early post-mitotic progenies.
The distribution of p53 mRNA in embryonic and neonatal
(P0) nervous system was grossly similar to that of Hes6 mRNA,
although p53 mRNA expression was less restricted to the
nervous system. On P7, expression of p53 mRNA was reduced
in the cerebral cortex (Ctx) while Hes6 mRNA expression was
maintained until this stage. p53 mRNA expression was not
detected in the adult brain except the adult dentate gyrus,
similarly to the expression pattern of Hes6 in the adult brain.
2.2.Effectof Hes6 overexpressiononthe survival ofneurons
Next, the effects of Hes6 overexpression on the neuronal
survival were explored following transfection of YFP-tagged
Hes6 or its mutants in the primary cultured cortical neurons
(Fig. 2). Previously we demonstrated that HeLa cells exhibit
Fig. 1 – DistributionofHes6(A,C,E,G)andp53(B,D,F,H)mRNA
in E16 (A, B) embryo and P0 (C, D), P7 (E, F), and adult (G, H)
brains. Embryos were parasagitally sliced prepared, and
postnatal brains were horizontal cut. Abbreviations are ctx,
cerebral cortex; CP, cortical plate; VZ, ventricular zone; Vt,
ventricle; Th, thalamus; Lv, liver; Pvr, paraventricular region;
Hip, hippocampus; Cbll, cerebellum; DG, dentate gyrus of
hippocampal formation.
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nuclearspeckle-like distribution of Hes6 whichwas associated
with PML nuclear body (Eun et al., 2008). On the other hand, in
neurons the speckle forms of Hes6-YFP signal were scattered
in the cytoplasm as well as in the nucleus (Fig. 2A). Similarly,
immunoreactivity of endogenous Hes6 was also found in the
cytoplasm and nucleus of primary cultured neurons (Fig. 2G).
Interestingly, mutant Hes6 lacking ability to associate with
PML nuclear body, ΔWRPW, failed to form nuclear and
cytoplasmic speckles (Fig. 2B; (Eun et al., 2008)), whereas
mutant Hes6 lacking basic domain (ΔBasic) which can interact
with PML nuclear body formed speckles in both nucleus and
cytoplasm, raising a possibility that both nuclear and cyto-
plasmic speckles in neurons may be related to the PML
association. Following transfection, a substantial proportion
of cells expressing wild-type or mutant Hes6 exhibited neurite
fragmentation, nuclear condensation (or fragmentation), and
cell death in cultured rat primary cortical neurons within 24 h
after transfection (Figs. 2D–F). Quantification of surviving
neurons among transfected (i.e. neurons exhibiting YFP
signal)demonstrated thatwild-typeandmutant Hes6resulted
in the less than ∼50% of Hes6+ cell survival (Fig. 2H).
Degenerating neurons following transfection of these Hes6
mutants including ΔWRPW all exhibited speckle-like aggrega-
tion of Hes6 signal widespread in the cytosol (Figs. 2D–F).
2.3.
neuronal death
Nuclear Hes6 is sufficient for the induction of
Because Hes6 localizes in both the nucleus and cytoplasm, it
was difficult to discriminate whether nuclear, cytoplasmic or
both forms of Hes6 are required for the induction of neuronal
death. To assess this issue, we further modified Hes6 and its
mutants to exclusively localize in the nucleus by fusing
nuclear localization signal (NLS) on its N-terminus (Figs. 3A–
F). Addition of NLS successfully targeted Hes6 or its mutants to
the nucleus: NLS-Hes6 (Fig. 3B), and NLS-ΔBasic (Fig. 3C)
formed nuclear speckles, whereas NLS-ΔWRPW (Fig. 3D)
exhibited diffused nuclear localization. Interestingly, all
these NLS-tagged Hes6 mutants exhibited cell death-inducing
activity (Fig. 3E). We also found that both Hes6 and ΔWRPW
can promote cell death in HeLa cells where they are not
localized in the cytoplasm (Fig. 3F; (Eun et al., 2008)).
Quantification of cell death of transfected cells demonstrated
that ΔWRPW as well as Hes6 exhibited apoptogenic activity
under the extended culture for 48 h. These results suggest that
the nuclear Hes6 is sufficient for the induction of cell death.
2.4.
cell death
Hes6 is involved in the Bax- and p53-dependent
Finally, we asked whether p53 and pro-apoptotic gene Bax are
required for the Hes6-induced neuronal death. We found that
the Hes6-induced dying neurons exhibited punctuated forms
of Bax-immunoreactivity (Fig. 4A), and cleaved caspase-3
immunoreactivity which are hallmarks for apoptotic cell
death. Furthermore, transient overexpression of Hes6 failed
to induce apoptosis of Bax−/−neurons whereas WT cortical
neurons underwent significant apoptosis (Fig. 4B). Interest-
ingly, cells expressing Hes6 or ΔWRPW appeared to exhibit
stronger cytoplasm p53 immunoreactivity, comparing to the
control cells (Fig. 4C). In neuronal processes, the p53 immu-
noreactivity was closely associated with cytoplasmic Hes6 or
ΔWRPW puncta. Furthermore, neurons derived from p53−/−
mice were also resistant to the overexpression of Hes6 and its
mutants (Fig. 4D), also supporting the idea that p53-Bax
pathway is critical for the Hes6-induced neuronal death. We
excluded the possibility that compensatory responses in the
absence of p53 gene are responsible for this resistance for the
neuronal death, because re-introduction of p53 into p53−/−
neurons restored the vulnerability of p53−/−neurons to Hes6
overexpression (Fig. 4E).
It is known that a pro-apoptotic function of p53 is mediated
by the transcriptional induction of pro-apoptotic genes such
as Bax and BH3-only proteins PUMA and Noxa (Lowe et al.,
2004). Therefore, to further evaluate the involvement of p53 in
the Hes6-induced apoptosis, we tested whether Hes6 over-
expression can enhance the expressions of these pro-apopto-
tic genes (Fig. 5). Because the efficiency of neuronal
transfection was too low (1–5%) to employ quantitative assays,
we transfected Hes6 plasmids to HeLa cells, and we found that
Bax and Noxa mRNA levels were increased 48 h after
transfection in a Hes6 dose-dependent manner. Collectively,
Fig. 2 – Cytoplasmic localization of Hes6 and the induction of
rat cortical neuronal death by Hes6 overexpression.
Following transient transfection of YFP-tagged Hes6 (A, D),
#WRPW (B, E), and #Basic (C, F) mutants in neurons, YFP
signals were observed as speckles (A, C) or diffused (B) form
in the nucleus and cytoplasm. Among the YFP-expressing
cells, a subset was degenerated (D–F). (G) Distribution of
endogenous Hes6 immunoreactivity in cultured cortical
neurons (DIV4). (H) Quantification of the transfected cell
viability. Data were expressed as mean±SEM, n=4. *P<0.05
in t-test comparisons with control YFP transfection group.
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these results demonstrated that Hes6 or its deletion mutants
induced neuronal death via p53- and Bax-dependent pathway.
3.Discussion
In this study, we found that overexpression of Hes6 exhibited
apoptosis-inducing activity in post-mitotic neurons. This
apoptogenic activity of Hes6 does not appear to be mediated
by the known physical or functional interaction with CBP or
PML (Eun et al., 2008), but requires p53- and Bax-dependent
apoptotic signal activation. Although the exact mechanism is
required to be elucidated, these results raise a possibility that
Hes6 is involved in the neuronal death induced by p53 during
development or in neurodegenerative diseases.
Hes6 is expressed in neural stem/progenitor cells in the
ventricular zone(VZ) andthe early post-mitotic neuronsin the
cortical plate of the perinatal brain (Pissarra et al., 2000;
Vasiliauskas and Stern, 2000). The distribution of p53 mRNA
was strikingly similar to that of Hes6 mRNA in the developing
and adult brain, suggesting that these two gene products may
function cooperatively. Considering that Hes6 acts synergis-
tically with p53 for cell cycle arrest, it is reasonable that they
localize together in proliferating neural progenitor cells. Cell
cycle control and cellular differentiation machineries have a
substantial crosstalk, and deficiencies in the cell cycle
regulators such as p53 and p21 affect the stem cell prolifer-
ation in mice (Cheng et al., 2000; Jacobs et al., 2006). The
expression of Hes6 in neural lineage stem/progenitor cells is
reported to promote neuronal differentiation and to suppress
glial differentiation partially via activation of neurogenin (Bae
et al., 2000; Gratton et al., 2003; Jhas et al., 2006; Koyano-
Nakagawa et al., 2000; Suzuki et al., 2003). While the direct
contribution of p53 to the differentiation of neural stem cells is
less clear, p53 activity is high in differentiating neuronal
precursors (Armstrong et al., 1995; Eizenberg et al., 1996; Sah et
al., 1995; Vaghefi et al., 2004). Furthermore, olfactory bulb stem
cells derived from p53−/−mice exhibited enhanced prolifera-
tion and favored differentiation into neurons (Armesilla-Diaz
et al., 2009; Medrano and Scrable, 2005; Meletis et al., 2006),
suggesting the role of p53 in the differentiating neural stem/
progenitor cells. In addition to the proliferating stem/progen-
itor cells, we also observed the expression of Hes6 and p53 in
early post-mitotic neurons, suggesting their roles in other
than the regulation of proliferation vs. differentiation of
proliferating cells. In post-mitotic neurons, p53 is an impor-
tant mediator of the apoptosis induced by irradiation, ROS,
and other neurotoxic reagents implicated in the neurodegen-
erative diseases (Culmsee and Mattson, 2005; Vila and
Przedborski, 2003). Other lines of evidence also link Hes family
to the control of apoptosis: Hes is one of the most prominent
downstream regulators of Notch signaling which is important
for the control of size and shape of the nervous system via the
regulation of cell death (Jehn et al., 1999; Miller and Cagan,
1998). To this end, we asked whether Hes6 can influence the
p53-dependent neuronal apoptosis.
Supporting above idea, ectopic expression of Hes6 in
primary cortical neurons evoked apoptotic death with an
activation of Bax- and p53-dependent pathways. We observed
that genetic deletion of p53 completely rescued neurons from
Hes6-induced apoptosis, suggesting that p53 is essential for
the execution of Hes6-induced apoptosis. p53 mediates
apoptosis by inducing the expression of pro-apoptotic genes
such as Bax and the BH3-only protein PUMA and Noxa that
cause mitochondrial membrane permeabilization (Jeffers et
al., 2003). Therefore, we initially assumed that apoptogenic
Fig. 3 – The effect of nuclear restriction of Hes6 and its mutants on rat cortical neurons. Comparing to wild-type Hes6 (A),
addition of nuclear localization signal (NLS) to Hes6 (B), #Basic (C), and #WRPW (D) resulted in the nuclear restriction of these
proteins. (E) Quantification of the transfected cell viability. Data were expressed as mean±SEM, n=4. *P<0.05 in t-test
comparisons with control YFP transfection group. (F) Quantification of viable cells 24–48 h after transfection of Hes6 or #WRPW
in HeLa cells. Data were expressed as mean±SEM, n=4. *P<0.05 in t-test comparisons with control YFP transfection group.
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activity of Hes6 is mediated by the modulation of p53′s
transcriptional activator function, and in fact we observed
that the overexpression of Hes6 can promote the expressionof
these pro-apoptotic genes. However, some of our results do
not support above idea. Previously we identified that Hes6
activates p53 activity via stimulation of CBP via its basic
domain (Eun et al., 2008), and deletion of CBP binding domain
of Hes6 (ΔBasic) attenuated cell cycle arrest activity of Hes6.
Because these interactions occur at PML nuclear body,
deletion of WRPW domain at C-terminus of Hes6 also
Fig. 4 – Apoptogenic function of Hes6 is mediated by Bax and p53-dependent pathway. (A) Cells undergoing apoptosis by Hes6
transfection exhibited Bax puncta (upper panel, arrow) and activated caspase-3 immunoreactivity (lower panel, arrow). (B)
Quantification of dying cells in Bax+/+(closed bar) or Bax−/−(open bar) cortical neurons transfected with Hes6 or control YFP
vectors. Data were expressed as mean±SEM, n=3. *P<0.05 in t-test comparisons. (C) Immunofluorescence labeling of p53 in
Hes6 (left panels) or #WRPW (right panels)-expressing cortical neurons. Note that p53 immunoreactivity was observed in the
cytoplasm of transfected cells. Large magnification view of the neurites (insets on the right of each image) shows the close
apposition of p53 immunoreactivity and Hes6 or #WRPWpuncta. (D) Quantification of viable cells in p53+/+(closed bar) or p53−/−
(open bar) neurons transfected with Hes6 or its mutants. Data were expressed as mean±SEM, n=3. *P<0.05 in t-test
comparisons. (E) Re-introduction of p53 into p53−/−neurons restored Hes6-induced neuronal death. Data were expressed as
mean±SEM, n=3. *P<0.05 in t-test comparisons.
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