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BioMed Central
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Neural Development
Open Access
Research article
Elevated P75NTR expression causes death of engrailed-deficient
midbrain dopaminergic neurons by Erk1/2 suppression
Kambiz N Alavian1,2, Paola Sgadò1,3, Lavinia Alberi1,4,
Srinivasa Subramaniam1,5 and Horst H Simon*1
Address: 1Interdisciplinary Centre for Neuroscience, Department of Neuroanatomy, Ruprecht-Karls-Universität, 69120 Heidelberg, Germany,
2Harvard Medical School, Neuroregeneration Labs, MRC 1, McLean Hospital, Mill St, Belmont, MA 02478, USA, 3Paola Sgadò, Neurogenetics
Laboratory, Child Neurology Unit, Pediatric Hospital A Meyer, Piazza di Careggi, 50139 Florence, Italy, 4The Johns Hopkins Institute for Cell
Engineering, Department of Neurology, North Broadway Street, BRB 720, Baltimore, MD 2120, USA and 5Department of Neuroscience, Johns
Hopkins Medical School, N Wolfe Street, Baltimore, MD 21210, USA
Email: Kambiz N Alavian - kambiz.alavian@gmail.com; Paola Sgadò - paola.sgado@gmail.com; Lavinia Alberi - lalberi2@jhmi.edu;
Srinivasa Subramaniam - ssubram9@jhmi.edu; Horst H Simon* -hsimon@mac.com
* Corresponding author
Abstract
Background: The homeodomain transcription factors Engrailed-1 and Engrailed-2 are required for
the survival of mesencephalic dopaminergic (mesDA) neurons in a cell-autonomous and gene-dose-
dependent manner. Homozygote mutant mice, deficient of both genes (En1-/-;En2-/-), die at birth
and exhibit a loss of all mesDA neurons by mid-gestation. In heterozygote animals (En1+/-;En2-/-),
which are viable and fertile, postnatal maintenance of the nigrostriatal dopaminergic system is
afflicted, leading to a progressive degeneration specific to this subpopulation and Parkinson's
disease-like molecular and behavioral deficits.
Results: In this work, we show that the dose of Engrailed is inversely correlated to the expression
level of the pan-neurotrophin receptor gene P75NTR (Ngfr). Loss of mesDA neurons in the Engrailed-
null mutant embryos is caused by elevated expression of this neurotrophin receptor: Unusually, in
this case, the cell death signal of P75NTR is mediated by suppression of Erk1/2 (extracellular-signal-
regulated kinase 1/2) activity. The reduction in expression of Engrailed, possibly related to the
higher levels of P75NTR, also decreases mitochondrial stability. In particular, the dose of Engrailed
determines the sensitivity to cell death induced by the classic Parkinson-model toxin MPTP and to
inhibition of the anti-apoptotic members of the Bcl-2 family of proteins.
Conclusion: Our study links the survival function of the Engrailed genes in developing mesDA
neurons to the regulation of P75NTR and the sensitivity of these neurons to mitochondrial insult.
The similarities to the disease etiology in combination with the nigral phenotype of En1+/-;En2-/-
mice suggests that haplotype variations in the Engrailed genes and/or P75NTR that alter their
expression levels could, in part, determine susceptibility to Parkinson's disease.
Published: 16 March 2009
Neural Development 2009, 4:11 doi:10.1186/1749-8104-4-11
Received: 18 November 2008
Accepted: 16 March 2009
This article is available from: http://www.neuraldevelopment.com/content/4/1/11
© 2009 Alavian et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Neural Development 2009, 4:11 http://www.neuraldevelopment.com/content/4/1/11
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Background
Mesencephalic dopaminergic (mesDA) neurons are the
main source of dopamine in the mammalian central nerv-
ous system. They are located in three distinct nuclei, sub-
stantia nigra pars compacta, ventral tegmentum and
retrorubral field. Their main innervation targets are the
basal ganglia, where they play key roles in the control of
emotion, motivation and motor behavior, documented
by their connection to schizophrenia, addiction and, most
prominently, to Parkinson's disease (PD) [1,2]. The main
characteristic of PD is the slow progressive loss of
dopaminergic neurons in the substantia nigra pars com-
pacta, causing diminished release of dopamine in the cau-
date putamen and debilitating motor deficits. Although
the molecular causes for the selective vulnerability of this
neuronal population are hardly understood, multiple
lines of evidence suggest mitochondrial dysfunction as a
major contributing factor [3] and apoptosis as the execu-
tioner of cell death [4]. Several mutations associated with
familial forms of PD encode mitochondrial proteins [5]
and neurotoxins specific to the nigrostriatal system –
MPTP (1-methyl-4-phenyl-,1,3,6-tetrahydropyridine), 6-
hydroxydopamine and rotenone – cause mitochondrial
damage as inhibitors of complex-I of the electron trans-
port chain [6]. Mitochondrial insult can cause oxidative
stress by production of reactive oxygen species, leading to
increased permeability of the mitochondrial membrane,
release of pro-apoptotic molecules, including cyto-
chrome-C, into the cytoplasm and, subsequently, to acti-
vation of caspases and induction of apoptosis [7].
Neuronal cell death can be a result of neurotrophin defi-
ciency. Action of the neurotrophins, consisting of nerve
growth factor (NGF), brain-derived neurotrophic factor
(BDNF), neurotrophin (NT)4/5 and NT3, is mediated via
a set of specific tyrosine kinase (Trk) receptors (TrkA, B, C)
and a common receptor, P75NTR (Ngfr). While the Trk
receptors signal survival, P75NTR can relay a survival or cell
death signal, depending on the cellular context and the
molecular form of the ligand [8]. The pro-survival func-
tion of neurotrophins and their receptors has been mainly
attributed to the downstream effect of phosphotidyl inosi-
tol-3 kinase (PI3K) and the extracellular-signal-regulated
kinase 1/2 (Erk1/2) pathways [9], whereas cell death sig-
naling via P75NTR is mediated by phosphorylation of c-Jun
N-terminal kinase (JNK) and BH3-only members of the
Bcl-2 family [10].
The homeodomain transcription factors Engrailed-1 (En1)
and Engrailed-2 (En2) are required for the survival and
maintenance of mesDA neurons in a cell-autonomous
and gene-dose-dependent manner, demonstrated by in
vitro cell mixing experiments, RNA interference (RNAi)
and analysis of chimeric mice [11-13]. In Engrailed double
mutant mice (En1-/-;En2-/-; from here on EnDM), the
mesDA neurons are generated and begin to express their
neurotransmitter phenotype but then die by apoptosis
between embryonic day (E)12 and E14, the stage when
Engrailed expression starts in their wild-type counterparts
[11,12]. The intermediate genotypes between EnDM and
wild type show various degrees of cell loss in the mesDA
system. Most interestingly, mice heterozygous null for En1
and homozygous null for En2 (En1+/-;En2-/-; from here
on EnHT), which are viable and fertile, exhibit a slow pro-
gressive loss of nigral dopaminergic neurons within the
first two months after birth, resulting in diminished stor-
age and release of dopamine in the striatum and in PD-
like motor deficiencies [14].
We show here that Engrailed-deficiency in mesDA neurons
leads to elevated P75NTR expression that is causal for cell
death if the neurons are null for En1 and En2. The death
signal is mediated by the suppression of Erk1/2 activity.
Probably linked to the P75NTR elevation, the dose of the
Engrailed genes also determines the sensitivity to mito-
chondrial insult.
Materials and methods
Animals
The generation of the En2 null mutant and the En1tau-
LacZ 'knock-in' mice have been previously described
[15,16]. The En2 mutants with an original mixed genetic
background of 129 and Swiss Webster were crossed three
times into a C57/BL6 background. The line was bred as
En1+/tlZ;En2-/- at the central animal facility, University of
Heidelberg.
Quantitative RT-PCR
Quantitative RT-PCR reactions were performed according
to the manufacture in a 7000 Sequence Detection System
from Applied Biosystems (Foster City, CA, USA) using
pre-formulated 'assays on-demand' and calculating the
results with the comparative cycle time (CT) method. The
pre-formulated 'assays on demand' had the following
identification tag: Mm00446294_m1 for Ngfr (P75NTR);
as standard control Mm00507222_s1 ribosomal protein
S18; Mm00435617_m1 for phosphoglycerate kinase 1
(Pgk1); and Mm00446973_m1 for TATA box binding
protein (Tbp). The dissected ventral midbrains were
homogenized, the RNA isolated and reverse transcribed
using random hexamers to initiate transcription. Each of
the individual PCR reactions was done in triplicate and at
least two of three standard controls were run in parallel.
Each of the experimental sets consisted of RNA from dif-
ferent pools of mutant and littermate control as well as
RNA from Tet-On induced Engrailed expressing N2A cells
and control.
Cell culture
All primary cell cultures were performed using E12.5
mouse embryos. EnDM embryos were distinguished from
the other genotypes in the litter by their midbrain/hind-
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brain morphology [11], which was occasionally verified
by PCR. Distinction between En2-/- and En1+/tlZ;En2-/-
embryos was achieved by incubation of the limb buds in
X-Gal solution at 37°C (40 mg/ml X-Gal (Sigma-Aldrich;
Munich, Germany) in 5 mM K3Fe(CN), 5 mM
K4Fe(CN)6x3H2O, 1 mM MgCl2 in phosphate-buffered
saline). For the cell culture, the neural tubes were dis-
sected and ventral midbrains were isolated. The tissue was
then dissociated using trypsine (Invitrogen; Karlsruhe,
Germany). The preparation of laminin (Sigma) coated
coverslips was described elsewhere [11]. The medium was
DMEM-F12 supplemented with 5% fetal calf serum,
0.25% bovine serum albumin (Sigma), 33 mM glucose,
50 U/ml penicillin, 50 U/ml streptomycin, and 1% Fung-
izone (Invitrogen). The cells were seeded at approximately
150,000 per cover slip and incubated at 37°C. After 36
hours, EnDM mutant cells were fixed in 4% paraformalde-
hyde and processed for immunostaining. The typical
numbers of tyrosine hydroxylase (TH)-positive cells per
cover slip were between 100 and 300 cells if wild-type or
heterozygote mutant tissue was dissociated. The numbers
of TH-positive neurons was always significantly lower if
the dissociated ventral midbrains were derived from E12
EnDM embryos. To obtain comparable numbers for
mutant and wild-type experiments, all cell counts were
normalized against each of the controls. Numbers pre-
sented in the results section (n) refer to the number of
experiments. Each of the experimental conditions is repre-
sented by at least three cover slips in each experiment. The
optimum concentration for the toxic substances was
determined by titration and checking for an intermediate
rate of survival (between 30% and 60%) in cultures of
mixed genotypes (En2-/- and En1+/-;En2-/-). For induc-
tion of cell death, serum was withdrawn from the medium
after 48 hours and the cultures were treated with the com-
pounds HA14-1, chelerythrine chloride (Axxora, San
Diego, CA)), prima-1, Apoptosis Activator-2, tumor
necrosis factor (TNF)α and 1-methyl-4-phenylpyridinium
(MPP+). Used concentrations, solvents, durations of treat-
ment and vendor sources are provided in Additional file
1[17-33]. The number 'n' corresponds to the number of
individual experiments conducted.
Design and transfection of siRNA oligos
The design of the 21-mer RNAi oligonucleotides was car-
ried out at Biomers.net (Ulm, Germany) and in accord-
ance with the protocol by Ebashir et al. [34]. Both RNA
duplexes targeted the coding sequence of P75NTR (A, sense
ACAGAACACAGUGUGUGAA(dTdT) and anti-sense
UUCACACACUGUGUUCUGU(dTdT); B, sense CAUUC-
CGACCGCUGAUGUUCU(dTdT) and anti-sense AACAU-
CAGCGGUCGGAAUGUG(dTdT)). For coupling with
Penetratin-One (QBiogene; Strasbourg, France), the sense
strand was modified with a thiol group on the 5' end. Tris-
2-carboxyethylphosphine (TCEP; 1 μl; Pierce; Bonn, Ger-
many) was added to 224 μl of the small interfering RNA
(siRNA; stock solution, 900 μM) and incubated for 15
minutes at room temperature. Then, 25 μl of Penetratin-1
was added, mixed and incubated for 5 minutes at 65°C
following by 1 hour at 37°C. Penetratin-1 solution was
reconstituted to 2 mg/ml (≈0.8 mM) in sterile water. A
stock solution of 20 mM TCEP in sterile RNAse/DNAse
free water was made. The aliquots were frozen at -80°C.
The Penetratin-coupled siRNA oligos were heated to 65°C
for 15 minutes and 3 μl of the mix was dissolved in com-
plete growth medium. As controls, we used Penetratin-1
coupled double-stranded RNA oligos directed against
Maged1 (Nrage) (UAACUUGAAUGUGGAAGAG(dTdT)
and CUCUUCCACAUUCAAGUUA(dTdT)) and the ran-
domly generated Scramble I Duplex (Dpharmacon; Hei-
delberg, Germany) sense CAGTCGCGTTTGCGACTGG
and antisense CCAGTCGCAAACGCGACTG. Both had no
effect on the survival rate of control or EnDM mesDA neu-
rons. Alternatively, uncoupled double-stranded RNA oli-
gos with the same sequences were transfected using
HiPerfect (Qiagen; Hilden, Germany) in accordance with
the manufacturer's protocol.
Immunohistochemistry and western blot analysis
The immunohistochemistry and western blot analysis was
done according to the protocol described elsewhere [35].
All the phospho-specific antibodies were purchased from
Cell Signaling Technology, the neurotrophin and TH anti-
bodies from Chemicon (Molsheim, France) and the anti-
bodies for neurotrophin receptors from Santa Cruz
Biotechnology (Heidelberg, Germany).
Statistical analysis
Values are expressed as mean ± standard error. Differences
between means were analyzed by using a paired, two-
tailed Student t-test. All shown p-values are rounded up at
the third or fourth digit.
Results
Elevated P75NTR expression in absence of Engrailed genes
A genome-wide expression analysis, using microarrays on
En1 inducible N2A cell line, identified the NGF receptor
P75NTR as downstream of the Engrailed transcription fac-
tors (data not shown). To confirm the microarray, we
examined by quantitative RT-PCR the levels of P75NTR in
the cell line as well as in ventral midbrain tissue in rela-
tionship to En1 expression. As a result of En1 induction in
N2A cells, the endogenous expression of P75NTR decreased
by 10-fold (9.9 ± 1.9%, p < 0.001, n = 8). Likewise, the
ventral midbrain tissue, derived from control littermates
(En2-/-) expressed almost 2.5-fold less P75NTR (42% ±
14.0%, p < 0.001, n = 6) than EnDM mutants (Figure 1A).
The western blot analysis confirmed the latter results; ven-
tral midbrain from En2-/- and EnHT embryos contained
74.4 ± 4.4% (p < 0.001, n = 3) and 60.2 ± 8.8% (p = 0.002,
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n = 3) less P75NTR protein, respectively, than the same tis-
sue from EnDM littermates (Figure 1B,C).
Since P75NTR can mediate cell death in neurons [8], we
began to investigate whether its elevated expression is
causal for the death of mesDA neurons in EnDM embryos.
In order to functionally interfere with P75NTR, we applied
an activity-blocking antibody (Rex) [28] to primary ven-
tral midbrain cell cultures. This antibody increased the
survival rate from 7.5 ± 1.24% to 34.8 ± 4.6% (p < 0.001,
n = 6; Figure 1D). Furthermore, to lower P75NTR expres-
sion levels in the mutant neurons, we applied specific
Penetratin-coupled siRNA duplexes [36]; 72 hours after
transfection, the total P75NTR protein was reduced by 83.2
± 6.3% (p = 0.05, n = 3; western blot not shown) and the
survival rate increased from 7.5 ± 1.24% to 25.1 ± 2.1% (p
< 0.001 n = 16) (Figure 1D). These data suggested that ele-
vated expression of P75NTR is the direct cause of the induc-
tion of apoptosis in Engrailed-deficient mesDA neurons.
P75NTR mediates dual, opposing functions of cell survival
and death, controlled by the presence or absence of neu-
rotrophins. For the anti-apoptotic function, neuro-
trophins require their cognate Trk receptors as
heterodimerization partners for P75NTR [8]. In order to
assess a potential role of the Trk/P75NTR system during the
course of cell loss, we determined the expression of the
Trk-receptors in E12 mesDA neurons. TrkC and TrkB, but
not TrkA, were detectable by immunohistochemistry and
western blot at equal levels in wild type and EnDM mutants
(Figure 2A–G).
The up-regulation of P75NTR and the presence of Trk recep-
tors suggested that Engrailed deficiency introduces a neu-
rotrophin requirement to the E12 mesDA neurons that
cannot be satisfied at this age, since the neurotrophins
specific to TrkB and TrkC – that is, BDNF, NT4 and NT3 –
are not expressed in the E12 ventral midbrain as they are
in the adult (Figure 2H). To test this hypothesis, we
applied saturating concentrations of BDNF, NT4 and NT3
to ventral midbrain cultures. After 72 hours, 50.2 ± 2.9%
(p < 0.0001, n = 27), 42.3 ± 10.1% (p < 0.001, n = 9) and
26.0 ± 3.5% (p < 0.001, n = 9), respectively, of the other-
wise dying EnDM mesDA neurons were still present in the
cultures (Figure 2I). The addition of BDNF to the control
littermate cultures demonstrated that this was due to an
elevated survival rate and not attributable to a higher rate
of precursor cell proliferation (Figure 3L). As expected
from the lack of TrkA, application of its ligand, NGF, did
not change the survival rate significantly. To test the spe-
cificity of BNDF, NT3 and NT4, we applied glial cell line-
derived neurotrophic factor (GDNF), growth differentia-
tion factor (GDF)-15 and transforming growth factor
(TGF)-β to the mutant cultures, all known survival factors
for mesDA neurons [37-39]. Similar to NGF, none of
them prevented the death of the Engrailed-deficient
mesDA neurons (Figure 2I). Furthermore, the linear dose-
response trend-line of the survival effect of BDNF (Figure
Elevated P75NTR expression is causal for cell deathFigure 1
Elevated P75NTR expression is causal for cell death. (A) Quantitative RT-PCR of ventral midbrain tissue (VM) derived
from EnDM (En1-/-;En2-/-) and EnHT E12 embryos, and of En1-expressing N2A cells inducible by doxycycline (Dox). P75NTR
expression is inversely correlated with En1 expression levels in tissues and cell lines. (B, C) Western blot analysis of the ven-
tral midbrain tissue shows the same relationship between P75NTR protein levels and En1 expression. Each active En1 allele
decreases the P75NTR expression level (n = 3, p = 0.002). (D) Ventral midbrain cultures derived from EnDM and En2-/- embryos.
Silencing of P75NTR by double-stranded RNA oligos and application of P75NTR-inhibiting antibody (Rex) increases the survival
rate of EnDM mesDA neurons compared to untreated control (Ctl) or after treatment with scrambled RNA oligos (n ≥ 6, p <
0.01). Error bars indicate standard error.
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2J) indicated a Kd value of approximately 2.5 nM, which
corresponds with the reported affinity of BDNF for the
TrkB/P75NTRcomplex [40].
The survival-mediating role of Erk1/2
The survival function of neurotrophins has been mainly
attributed to Erk1/2 and PI3K pathways [41]. As the next
step, we examined whether these pathways play a role in
arbitrating the effect of the neurotrophins on Engrailed-
deficient mesDA neurons and whether they intersect with
the molecular events regulated by Engrailed expression.
Therefore, we studied activation of the two pathways,
using antibodies against the phosphorylated forms of
Erk1/2 and AKT [41]. Immunohistochemistry showed
that while total Erk1/2 protein was present in both En2-/-
and EnDM mesDA neurons, it was activated only in the
wild-type-like cells and not in EnDM mesDA neurons (Fig-
ure 3A–D). Western blot analysis of ventral midbrain tis-
sue showed a similar effect; Erk1/2 activity in E12 EnDM
ventral midbrain was reduced by 66.8 ± 18.2% (p = 0.02,
Loss of Engrailed induces neurotrophin requirement in mesDA neuronsFigure 2
Loss of Engrailed induces neurotrophin requirement in mesDA neurons. (A-F) Double immunohistochemistry on
dissociated cells derived from En2-/- (A-C) and En1-/-;En2-/- (EnDM) (D-F) E12 ventral midbrain using antibodies against tyrosine
kinase (Trk)B (A, D), TrkC (B, E), P75NTR(C, F) and TH (green) counterstained with DAPI. TrkB, TrkC and P75NTR are
expressed by TH+ cells from both genotypes; however, the immunohistochemistry is not sensitive enough to detect differ-
ences in P75NTR expression between genotypes. (G, H) Western blot of ventral midbrain tissue derived from different
Engrailed genotypes. The two Trk receptors do not depend on Engrailed expression (G). Brain-derived neurotrophic factor
(BDNF), neurotrophin (NT)4 and NT3 are not expressed in E12 ventral midbrain tissue, but they are in the adult (H). (I)
Treatments (>10 ng/ml) for 72 hours with TrkB/C-specific neurotrophins – BDNF, NT4 and NT3 – greatly increases the sur-
vival rate of EnDM mesDA neurons (n ≥ 6; p < 0.001), whereas nerve growth factor (NGF), glial cell line-derived neurotrophic
factor (GDNF), transforming growth factor (TGF)-β and growth differentiation factor (GDF)-15 do not significantly alter sur-
vival rate. (J) Dose response curve: BDNF concentration plotted against survival rate showing saturation at approximately the
10 ng/ml. Scale bars: 25 μm. Error bars indicate standard error. Ctl, control.
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Differential activation of Erk1/2 in mesDA neuronsFigure 3
Differential activation of Erk1/2 in mesDA neurons. (A-D, G-K) Immunohistochemistry of E12 ventral midbrain cell
culture stained against TH (green), total Erk1/2 protein (red) (A, B) and phosphorylated Erk1/2 (red) (C-D, G-K). (A-D) While
Erk1/2 protein is present in mesDA neurons of both genotypes (A, B), it is only phosphorylated in En2-/- mesDA neurons (C)
and not in the En1-/-;En2-/- (EnDM) counterparts (D). (E-I) Erk1/2 becomes activated in EnDM mesDA neurons after treatment
with the survival-inducing neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)4 and NT3, or after
silencing of P75NTR (RNA interference (RNAi)) (G-J), but not when glial cell line-derived neurotrophic factor (GDNF) is applied
(I). (E) Western blot of E12 ventral midbrain tissue confirms the immunohistochemical finding of differential phosphorylation
between genotypes and shows that neither AKT, part of the phosphotidyl inositol-3 kinase pathway, nor other mitogen-acti-
vated protein kinases, such as JNK and P38, are differentially activated. (F) Quantification of phosphorylated Erk1/2 in western
blot normalized against En2-/- tissue. (L) Number of TH-positive cells in EnDM and En2-/- ventral midbrain cultures after 72
hours, treated with the 400 nM Mek inhibitor U0126 in conjunction with BDNF, Penetratin-coupled P75NTR double-stranded
RNA oligonucleotides and the P75NTR inhibiting antibody (Rex). Numbers are normalized against untreated cultures at 24
hours. The rescue effect is significantly reduced when the EnDM cultures are treated with the Erk1/2 inhibitor. Scale bars: 25
μm. Error bars indicate standard error. Ctl, control.
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n = 3) in comparison to the same tissue derived from their
En2-/- littermates (Figure 3E,F). In contrast to Erk1/2, we
did not detect differential activation of AKT in the western
blot (Figure 3E). Additionally, we could not detect signs of
differential activation of other components of the
mitogen-activated protein kinase (MAPK) pathways, such
as JNK or P38 (Figure 3E).
The loss of Erk1/2 phosphorylation suggested that this
MAPK pathway was differentially activated in EnDM
mesDA neurons in response to a survival or death signal.
To investigate this hypothesis, we examined the state of
activation of Erk1/2 after application of the survival fac-
tors. The addition of BDNF, NT4 and NT3, and the knock-
down of P75NTR by siRNA oligonucleotides all caused
phosphorylation of Erk1/2 (Figure 3G–J); however,
MAPK was not activated after application of NGF, GDNF,
TGF-β or GDF-15 (for example, see Figure 3K). If the loss
of Erk1/2 activity is the primary cause of cell death, when
P75NTR is elevated in mesDA neurons, and this correlation
is not accidental, then inhibition of the Erk1/2 pathway
should interfere with the rescue by the neurotrophins and
by P75NTR inhibition or silencing. To test this, we concur-
rently treated the cultures with U0126 [42], an inhibitor
of the MAPK kinase upstream of Erk1/2, MEK1/2 [32], at
a concentration (400 nM) not toxic to the wild-type (En2-
/-) neurons. The inhibition of Erk1/2 by U0126 signifi-
cantly reduced the rescue effect of all three survival factors;
from 42.2 ± 3.1% to 11.7 ± 3.5% (p = 0.002) for BDNF,
from 34.7 ± 4.7% to 8.3 ± 2.7% (p < 0.0001) for Rex and
from 25.4 ± 2.7% to 10.8 ± 1.9% (p = 0.009) after P75NTR
silencing (control mutant 4.2 ± 2.2%, p = 0.01, n = 4 for
all experiments) (Figure 3L).
To elaborate further on the correlation between the
expression of P75NTR and the state of phosphorylation of
Erk1/2, we silenced the P75NTR expression in EnHT ventral
midbrain cultures by RNAi, using two methods with dif-
ferent transfection efficiencies (the lipophilic transfection
reagent HiPerfect, and Penetratin-coupled oligos). The
former reduced P75NTR expression levels, on average, by
63.3 ± 2.0% (p = 0.011, n = 3) and the latter by 82.2 ±
6.3% (p = 0.05, n = 3). This, in turn, caused 6.09 ± 1.40-
fold (p = 0.01, n = 3) and 8.64 ± 1.45-fold (p = 0.008, n =
3) increases, respectively, in the phosphorylation of Erk1/
2 (Figure 4A–D).
Sensitivity to mitochondrial dysfunction and Engrailed
expression level
P75NTR signaling often mediates cell death via induction
of the mitochondrial (intrinsic) pathway of apoptosis
[43,44]. To further assess whether the elevated level of
P75NTR expression in the Engrailed-deficient mesDA neu-
rons is causal for demise of the cells, we investigated the
dying neurons for signs of this pathway. We had previ-
ously reported that loss of Engrailed expression in mesDA
neurons causes activation of caspase-3 [11,14], an effector
caspase, triggered by the intrinsic or extrinsic pathways of
apoptosis [45,46]. The intrinsic death pathway involves
release of cytochrome C from the mitochondria, which
participates in formation of apoptosome, which is
required for activation of caspase-9. We detected small,
rounded TH-positive cells with active caspase-9, and
pyknotic, fragmented nuclei in the ventral midbrain of
Engrailed-deficient E13 embryos as well as in EnHT mice
during the postnatal stages of nigral cell loss (postnatal
day 20; Figure 5A–C) confirming our hypothesis that the
pathway downstream of P75NTR signaling must be the
cause of the demise of these neurons in the absence of the
Engrailed genes [43].
Erk1/2 activation is inversely correlated with P75NTR expres-sionFigure 4
Erk1/2 activation is inversely correlated with P75NTR
expression. (A, B) Immunohistochemistry on E12 dissoci-
ated ventral midbrain cultures derived from EnHT E12
embryos stained against TH (green) and P75NTR. P75NTR
expression is absent in cultures treated with Penetratin-cou-
pled double-stranded RNA oligos. (C) Western blot of ven-
tral midbrain cultures using two methods of RNA
transfection with different efficiencies: HiPerfect (HiP) and
Penetratin-coupled RNA oligos show increase in phosphor-
ylated Erk1/2 after silencing of P75NTR, but no changes in
phosphorylation of P38. (D) Quantification of P75NTR
expression and Erk1/2 phosphorylation after transfection
with P75NTR double-stranded RNA oligonucleotides, normal-
ized against untreated EnHT cultures, showing the inverse cor-
relation between the two parameters. Scale bars: 25 μm.
Error bars indicate standard error.
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P75NTR expression in tumor cells induces a dose-depend-
ent increase of the pro-apoptotic members (Bad, Bax and
Bak) and a decrease of the anti-apoptotic members (Bcl-2
and Bcl-XL) of the Bcl-2 family [47]. Since we were unable
to directly measure the relative amounts of these proteins
in mesDA neurons themselves, we followed up on this
possibility by application of HA14-1 and chelerythrine
chloride, inhibitors of Bcl-2 and Bcl-XL, respectively.
HA14-1 was discovered as an inhibitor of Bcl-2 using a
computer screening strategy based on its predicted struc-
ture [24]. Chelerythrine chloride was identified by a high-
throughput screening of natural compounds [48]. Both
are highly specific for their targets. If the levels of the two
anti-apoptotic members of the Bcl-2 family of proteins are
lowered, as suggested by these previous tumor cell-exper-
iments [47], the sensitivity to HA14-1 and chelerythrine
chloride should be higher when the level of Engrailed pro-
tein decreases. Twenty-four hours after treatment with
HA14-1 and chelerythrine chloride, the rate of survival of
mesDA neurons, derived from E12 En2-/- embryos, was,
on average, 75.3 ± 6.8% and 92.7 ± 2.7% (p < 0.001, n =
7 for both compounds) higher, respectively, than their
counterparts derived from EnHT littermates (Figure 6A,B),
suggesting that reduced Engrailed and elevated P75NTR
expression lowers the threshold at which the intrinsic
pathway of apoptosis is triggered.
These results could indicate that the reduction in Engrailed
expression increases the vulnerability of mesDA neurons
to mitochondrial dysfunction in a general manner, since
the three most commonly used reagents to model PD –
MPP+, 6-Hydroxydopamine (6-OHDA) and rotenone
[17,29,49,50] – also cause cell death by induction of this
pathway of apoptosis, evident from activation of caspase-
9 [51-53] (Figure 7A–C). To test this hypothesis directly,
we treated EnHT mesDA neurons with MPP+, the metabo-
lite of MPTP, an inhibitor of complex-I of the mitochon-
drial electron transport chain [49]. After 48 hours in
culture, the rate of survival of En2-/- mesDA neurons was,
on average, 124.5 ± 5.0% (p < 0.001, n = 12) higher than
for their En1 heterozygote (EnHT) littermates, suggesting
that MPTP and Engrailed may act upon the same molecu-
lar pathway upstream of caspase-9 (Figure 6A,B).
To determine whether this sensitivity to cell death is spe-
cifically related to instability of the mitochondria, we also
employed Apoptosis Activator-2 [19], Prima-1 [27], and
TNFα [31]. The first induces cell death by triggering apop-
tosome formation, the second by activation of p53 and
the third, as a ligand of the TNF receptors, induces the
extrinsic, receptor-mediated pathway of apoptosis. In all
three cases, the level of En1 expression did not have a sig-
nificant influence on the survival rates of these neurons (-
8.1 ± 6.0% (p = 0.67) for Apoptosis Activator-2, 4.6 ±
Mitochondrial apoptosis in Engrailed-deficient mesDA neuronsFigure 5
Mitochondrial apoptosis in Engrailed-deficient mesDA neurons. (A-C) TH and activated caspase-9 immunohistochem-
istry on ventral midbrain coronal sections of E13 En1-/-;En2-/- (EnDM) embryos (A), of En1+/-;En2-/- (EnHT) postnatal day 20
brain (C), and of EnDM E12 cell culture (B) counterstained with the nuclear marker DAPI. Dying TH-positive neurons (arrows)
exhibit small rounded cell bodies and signs of apoptosis, that is, activated caspase-9 and pyknotic nuclei (DAPI). (D) EnDM ven-
tral midbrain cultures treated for 72 hours with inhibitors for caspases-3, -8 and -9, a pan-caspase inhibitor, z-vad-fmk, the
BAX inhibitor Ku70, the P53 inhibitor Pfithrin-α, and the JNK inhibitor SP600125. None of the treatments significantly changed
the survival rate of EnDM mesDA neurons. The number of surviving TH-positive cells was normalized in each case against
untreated cultures 24 hours after dissociation. Scale bars: 25 μm. Error bars indicate standard error.
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6.2% (p = 0.82) for Prima-1, and 5.1 ± 3.1% (p = 0.48) for
TNFα; n = 7 for all three experiments; Figure 6A,B).
Gain of function in a mesDA cell line
To confirm the Engrailed dose-dependent sensitivity of
mesDA neurons to mitochondrial insult, we performed a
gain-of-function experiment using the inducible Tet-On
system to express En1 in the dopaminergic cell line MN9D
[54]. As in the primary cultures, En1 was protective against
cell death if induced by administration of MPP+ (42.4 ±
0.7%, p < 0.0001, n = 23), chelerythrine chloride (46.7 ±
1.3%, p < 0.0001, n = 10) and HA14-1 (32.4 ± 1.3%, p <
0.0001, n = 10). Accordingly, survival rate was not signif-
icantly altered if the other three reagents were employed (-
10.7 ± 4.7% (p = 0.26) for Apoptosis Activator-2, -8.3 ±
5.2% (p = 0.50) for Prima-1, and 1.0 ± 7.4% (p = 0.95) for
TNFα; n = 12 for all experiments; Figure 6C,D).
Functional inhibition of Bcl-2 and Bcl-XL and application
of MPP+ induces apoptosis by release of cytochrome C
from the mitochondrial inter-membrane space into the
cytosol [55,56]. The protective effect of En1 against HA14-
1, chelerythrine chloride and MPP+ may be attributable to
higher mitochondrial stability. To test this hypothesis, we
compared the cytosolic and mitochondrial protein frac-
tions of En1-expressing MN9D cells to non-expressing
cells. The proportion of cytochrome C in the mitochon-
dria was always significantly higher after induction of En1,
either in the presence of MPP+ or in untreated control cul-
tures (76.1 ± 7.3%, p = 0.03, n = 3, and 41.6 ± 7.0%, p =
0.005, n = 3, respectively), whereas the total amount of
cellular cytochrome C was unaltered (Figure 6F,H). In
contrast, the proportional levels in mitochondria and
cytosol of the apoptosis inducing factor, which causes cas-
pase-independent mitochondrial apoptosis [57], were
independent of the level of En1 expression, suggesting
that Engrailed participates in the regulation of mitochon-
drial stability via the cytochrome C/caspase-dependent
pathway of apoptosis rather than the caspase-independ-
ent pathway, represented by apoptosis inducing factor.
The sensitivity to induction of the intrinsic pathway of apoptosis correlates with En1 expressionFigure 6
The sensitivity to induction of the intrinsic pathway of apoptosis correlates with En1 expression. (A-D) Ventral
midbrain cultures 24 hours after application of apoptosis-inducing compounds. Charts of EnHT (En1+/-;En2-/-) and En2-/- E12
cultures depicting the number of surviving TH-positive cells (A, B) and cultures of En1-inducible MN9D cells depicting cell sur-
vival measured by cell proliferation assay (C, D). Surviving cells were normalized against untreated EnHT cultures (A) or
untreated non-induced MN9D cells (C), treated EnHT cultures (B) or treated non-induced MN9D cells (D). Higher Engrailed
expression reduced the cell death rate after MPP+, HA14-1 and chelerythrine chloride (CC) treatment, whereas the rate of cell
survival after application of the tumor necrosis factor alpha (TNFa), Prima-1 and Apoptosis Activator-2 (AA2) does not corre-
late with the level of En1 expression. Dox, doxycycline. (E) Western blot analysis of mitochondrial and cytoplasmic protein
fractions of MN9D cells 72 hours after En1 induction and 24 hours after MPP+ treatment. (F) Proportion of cytochrome C
(Cyt-C) in cytosol is lower in En1-expressing MN9D cells before and after MPP+ treatment. Scale bars: 25 μm. Error bars indi-
cate standard error. 6 ≤ n ≤ 27, *p ≤ 0.001, **p ≤ 0.0001. Cyt, cytosolic; Mit, mitochondrial; ANT, adenine nucleotide trans-
porter; Dox, doxycycline.
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Discussion
In this study, we provide evidence that cell death in
Engrailed-deficient mesDA neurons is a result of higher
P75NTR expression and the loss of Erk1/2 activity. Further-
more, we show that the dose of Engrailed is part of the
molecular mechanism that determines the sensitivity of
these neurons to mitochondrial insult.
P75NTR can cause apoptosis in various neuronal popula-
tions by mere high expression [10] or as a mediator of a
(pro-)neurotrophin death signal [8]. Alternatively, since
TrkB and C are expressed by the mesDA neurons, the
abnormal increase in the expression of P75NTR could
introduce a neurotrophin dependency, which does not
occur in the wild type at this age. The latter is more likely
in mesDA neurons deprived of the Engrailed genes, since
the death signal could be counteracted by addition of neu-
rotrophins (BDNF, NT3, NT4) [58]. Furthermore, the Kd
calculated from the dose response of BDNF, corresponds
to the known affinity of neurotrophins for P75NTR and Trk
receptors [40], demonstrating that the survival effect of
the neurotrophins is attributable to direct binding to the
receptors on the surface of mesDA neurons, as opposed to
a survival signal that originates from the surrounding
cells, which would be reflected in a different shape of the
dose response curve. A direct interaction of the neuro-
trophins with receptors on mesDA neurons is also consist-
ent with our previous findings that the Engrailed genes are
cell-autonomously required for the survival of these cells
[11]. The ineffectiveness of Ngf can be readily explained
by the lack of expression of TrkA. It is noteworthy that nei-
ther neurotrophins nor inhibition of P75NTR completely
(that is, 100%) rescue mesDA neurons deprived of
Engrailed genes, and it is thus possible that P75NTR is only
one of many factors contributing to the death of mesDA
neurons in the absence of the Engrailed genes. However,
since the in vitro timeframe of loss of EnDM mesDA neu-
rons is 72 hours, it is also possible that those cells, which
died anyway, had already been committed to cell death
before the knock-down of P75NTR by the RNA duplexes
was sufficiently high or the neurotrophin effect set in.
Neurotrophin-induced Trk/P75NTR interaction, or lack
thereof, can provoke intracellular activation of the MAPK
and PI3K pathways. Among MAPKs, JNK and p38 partici-
pate in stress responses and often trigger apoptosis, while
Erk1/2 signaling regulates cell proliferation, differentia-
tion and survival [59]. Alternatively, the pro-survival role
of neurotrophins can be mediated by PI3K signaling. This
is in contrast to our findings. The activity of JNK, p38 or
PI3K is independent of the Engrailed genes and the level of
P75NTR expression in mesDA neurons. The lack of Erk1/2
activity in EnDM mesDA neurons, which is reversed under
any of the rescue conditions, demonstrates that the level
of Erk1/2 phosphorylation in mesDA neurons is corre-
lated with Engrailed expression and inversely to the level
of P75NTR, suggesting that the P75NTR death signal is medi-
ated in these neurons by suppression of Erk1/2 activity.
Although the association of P75NTR and the Erk proteins
has been shown in PC12 cells [60], regulation of the activ-
ity of the Erk1/2 signaling pathway as a result of death sig-
naling by P75NTR is a novel mechanism, which signifies
both the role of sustained activity of Erk1/2 in the survival
and/or of P75NTR in the demise of mesDA neurons. Our
data also suggest that the survival effect of neurotrophins
may be the result of dis-inhibition of a death signal (high
expression of P75NTR) that is triggered in the absence of
the proper transcriptional regulation (by the Engrailed
genes) during the course of development or possibly
throughout life.
The pro-apoptotic role of P75NTR has been established in
the animal models of neurodegenerative disorders, other
than PD, including beta-amyloid peptide-dependent cell
death [61], or stress conditions, including ischemia [62].
Although function of the ligands and the co-receptors of
P75NTR, TrkA/B/C, have been investigated in mesDA neu-
rons [63-65], the role of P75NTR, itself, has not been
reported in this system. Our work provides the first evi-
dence for the pivotal role of P75NTR in mesDA neurons, as
a negative mediating factor for the lifelong survival func-
tion of the Engrailed genes in this neuronal population.
The sensitivity to neurotoxin-induced cell death by MPTP
and to inducers of mitochondrial instability, such as the
Bcl-2 and Bcl-XL inhibitors, is inversely correlated with the
dose of Engrailed expression in mesDA neurons. Mito-
chondrial dysfunction was first implicated in the patho-
Mitochondrial apoptosis after complex I inhibitionFigure 7
Mitochondrial apoptosis after complex I inhibition.
(A-C) Immunohistochemistry of E12 ventral midbrain cell
cultures. Caspase-9 is activated in wild-type mesDA neurons
(arrows), treated with MPP+, rotenone, or 6-OHDA.
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genesis of PD after accidental administration of MPTP by
drug abusers and a consequent Parkinsonian syndrome.
Lowered complex I activity and reduced mitochondrial
stability [3] are believed to be key factors in the etiology
of PD. In postmortem brains of PD patients, reduced
activity of complex-I (NADH/ubiquinone oxidoreduct-
ase) of the mitochondrial electron transport chain [49]
and elevated levels of the pro-apoptotic members of the
Bcl-2 family have been observed in the substantia nigra
[66]. Then, three genes, DJ1, PINK1 and OMI/HTRA2,
mutations of which have been associated with familiar
forms of PD, were discovered to have a function in the
mitochondria [5]. Furthermore, the toxicity of MPTP is
conferred by inhibition of complex I of the electron trans-
port chain, triggering the release of cytochrome-C from
the mitochondrial intermembrane space into the cytosol,
which involves Bcl-2 family members [7]. Here, we dem-
onstrate that the sensitivity to neurotoxin-induced cell
death and to inducers of mitochondrial apoptosis, such as
the Bcl-2 and Bcl-XL inhibitors, is inversely correlated to
the dose of Engrailed expression in mesDA neurons. This,
the previous findings of other groups, and the PD-like
slow progressive loss of nigral dopaminergic neurons in
EnHT mice [14] suggest that the mode of action of the
Engrailed genes converges with MPTP toxicity and possibly
also with the disease mechanism on the mitochondria.
Intriguingly, a recent association study into PD indicated
a single nucleotide polymorphism in the intron of En1 as
a potential risk factor for sporadic forms of this disease
[67].
Abbreviations
BDNF: brain-derived neurotrophic factor; E: embryonic
day; En: Engrailed; EnDM: Engrailed double mutant mice
(En1-/-;En2-/-); EnHT: heterozygous null for En1 and
homozygous null for En2 (En1+/-;En2-/-); Erk: extracellu-
lar-signal-regulated kinase; GDF: growth differentiation
factor; GDNF: glial cell line-derived neurotrophic factor;
JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated
protein kinase; mesDA: mesencephalic dopaminergic;
MPTP: 1-methyl-4-phenyl-,1,3,6-tetrahydropyridine;
NGF: nerve growth factor; NT: neurotrophin; PD: Parkin-
son's disease; PI3K: phosphotidyl inositol-3 kinase; RNAi:
RNA interference; siRNA: small interfering RNA; TGF:
transforming growth factor; Trk: tropomyosin-receptor-
kinase.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KNA and HHS conceived of, designed and discussed the
studies, carried out the experiments, analyzed and inter-
preted the data and wrote the draft and the final version
of the manuscript. PS and LA made the stable MN9D cell
lines. SS helped with immunoassays. The manuscript was
approved by all authors.
Additional material
Acknowledgements
This work was supported by grants from the Federal Secretary for Educa-
tion and Research BMBF Biofuture 98, the German Research Council SI
752/3-1 and the Michael J Fox Foundation. We thank Martyn Goulding for
the En1/tauLacZ, Alex Joyner for the En2 mutant mice, Louis Reichardt for
the Rex antibody, and Jutta Fey for technical assistance.
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