Department of Neuroanatomy, Interdisciplinary Center of Neuroscience, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany
* Author for correspondence (e-mail: horst.simon{at}urz.uni-heidelberg.de)
Accepted 17 February 2004
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SUMMARY |
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Key words: Transcription factors, Ventral tegmentum, Neurodegenerative disease, Neuronal survival, Neuronal differentiation
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Introduction |
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The engrailed (En) genes are involved in regionalisation during early
embryogenesis (Hidalgo, 1996;
Joyner, 1996
), and later in
the specification of certain neuronal populations
(Lundell et al., 1996
;
Simon et al., 2001
). In
mammals, two homologues of En have been identified, En1 and
En2. They are both expressed by all mesencephalic DA neurons (mDA)
from early in development into the adult
(Simon et al., 2001
).
Homologous recombinant mutant mice null for En1 and En2 show
a large deletion in the midbrain and anterior hindbrain
(Liu and Joyner, 2001
;
Simon et al., 2001
). Despite
this deficiency, the mDA neurons are generated, become postmitotic and express
tyrosine hydroxylase (Th), the rate-limiting enzyme of dopamine
synthesis. However, soon thereafter, the cells disappear, and at P0 the entire
mDA system is absent.
The large deletion of mid-hindbrain tissue in the mutant raises the question whether the surrounding neuroepithelium provides essential support or whether the En genes are cell-autonomously required for the survival of mDA neurons. We have addressed this issue using in vitro cell mixing experiments and RNA interference technologies. We show that the En genes are cell-autonomously required for the survival of mDA neurons and that the loss of En expression in mDA neurons induces apoptosis with a time course of less than 24 hours. These findings may open the paths to novel molecular links to PD.
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Materials and methods |
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Primary cell culture
All primary cell cultures were performed using E12.5 mouse embryos. En
double embryos were distinguished from their littermate counterparts by their
midbrain/hindbrain morphology (16), which was occasionally verified by PCR.
The embryonic neural tubes were dissected carefully removing meninges,
followed by isolation of the ventral midbrains. The tissue was then
dissociated using accutase (PAA Laboratories, Germany). The preparation of
laminin-(Sigma, Germany) coated, membrane vesicle-coated and 3-D collagen
matrix cultures are all described elsewhere
(Krieglstein et al., 1995;
Wizenmann et al., 1993
;
Yee et al., 1999
). The medium
was DMEM-F12 supplemented with 10% horse serum (HS), 5% fetal calf serum
(FCS), 33 mM glucose, 50 U/ml penicillin and 50 µg/ml streptomycin. The
cells were seeded at a concentration of 150,000 per well or coverslip and
incubated from 1 hour to 5 days. In the cell mixing experiment, cell numbers
of mutant and wild type were each 75,000 cells. For the RNA interference, we
used always serum-free medium DMEM-F12 medium containing 1-2
supplement (Gibco), 33 mM glucose, 50 U/ml penicillin and 50 µg/ml
streptomycin.
Immunohistochemistry
Cultured cells and all tissues were fixed with 4% paraformaldehyde in 100
mM phosphate buffer (pH=7.4). All immunostaining was performed as described
(Simon et al., 2001) using
rabbit and sheep anti-TH antibodies (AB152 and AB1542 Chemicon, Germany) at
1:1000, rabbit anti-activated caspase 3 (Catalog #9661 Cell Signaling, USA) at
1:500, rabbit anti-Pbx1/2/3 (sc888 Santa Cruz, USA) at 1:2000, mouse
monoclonal anti-Engrailed 4g11 (Developmental Studies Hybridoma Bank) pure
supernatant, and mouse monoclonal anti-BrdU (Catalog #1170376 Roche
Diagnostic, Germany) at 1:50. Biotinylated or directly coupled
species-specific antibodies were all obtained from Jackson Immuno Research,
USA. The antibodies or the streptavidin were conjugated with Cy2, Cy3, Cy5 or
horseradish peroxidase. After immunostaining, tissue and cultured cells were
counterstained with DAPI (Catalog #236276 Roche, Diagnostic).
siRNA design and transfection
The siRNA duplexes were designed as described elsewhere
(Elbashir et al., 2001b). In
brief, the position of the 21 nucleotide siRNA duplexes were chosen at least
150 nucleotides 3' to the first ATG of the coding region. The sense and
antisense of each duplex were complementary at 19 nucleotides and had a
two-nucleotide overhang at the 3' terminus. We used the following RNA
nucleotides for the experiments: En1a (NM_010133),
CAUCCUAAGGCCCGAUUUCTT (sense) and GAAAUCGGGCCUUAGGAUGTT (antisense);
En1b, GUUCCCGGAACACAACCCUTT (sense) and AGGGUUGUGUUCCGGGAACTT
(antisense); lamin A/C (NM_019390), GCAGCUUCAGGAUGAGAUGTT (sense) and
CAUCUCAUCCUGAAGCUGC (antisense); Pbx1 (AF020196)
CAGUUUUGAGUAUUCGGGGTT (sense) and CCCCGAAUACUCAAAACUGTT (antisense); and the
randomly generated Scramble I Duplex (Dpharmacon, USA), CAGTCGCGTTTGCGACTGG
(sense) and CCAGTCGCAAACGCGACTG (antisense). All RNA duplexes were purchased
from MWG-Biotech, Germany. Three to five days after the dissociation, RNA
oligo transfection was performed using Transmessenger transfection reagent
(Qiagen, Germany). siRNA (0.1-0.3 µg per well) was condensed with 0.6 µl
Enhancer R in 50 µl Transmessenger buffer, and complexed with 1.5 µl of
Transmessenger reagent. The transfection complex was diluted in 500 µl
DMEM/F12 1-2 supplement, then added to the cells, 2 hours post-transfection
the medium was replaced with fresh complete medium as described above.
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Results |
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Engrailed and axonal outgrowth
The death of mDA neurons in the En double mutant embryo might be related to
the absence of axonal outgrowth, as the cells may lack essential molecular
support from their innervation target. Involvement of En in axonal outgrowth
has been demonstrated in mammals and insects
(Marie et al., 2002;
Saueressig et al., 1999
). To
address this possibility, we placed dissociated E12 ventral midbrain into a
three-dimensional collagen matrix with or without an explant of basal
telencephalon of the same age. Alternatively, we used glass coverslips coated
with laminin or membrane vesicles derived from E12 midbrain
(Fig. 2A-C). During the first
24 hours in culture, the mutant mDA neurons differentiated normally. They
acquired a spindle form shape and extended neurites. The mean length of their
processes matched exactly that seen in the littermate controls (mixture of
En1+/;En2/ and
En2/)
(Fig. 2D). However, cell death
was only postponed, and after a further 48 hours of incubation almost all of
the mutant DA neurons disappeared, regardless of culture condition
(Fig. 2E, for survival of
control cells see Fig. 3). The
majority of the cells died between 24 and 48 hours after dissociation, showing
the same signs of apoptotic cell death we observed on tissue sections,
activation of caspase 3 and appearance of pyknotic nuclei
(Fig. 2F-I). In the littermate
control cultures, the loss of TH-positive cells after 72 hours in culture is
not higher than 10% to 15% (see Fig.
3G for control experiment). These findings demonstrate that the
lack of DA axonal outgrowth in En double mutants is not the reason for the
cell death and that the mutant mDA neurons are viable and differentiate
normally, until a requirement for the En genes sets in, which then becomes
essential for their survival.
|
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Rapid induction of cell death after ablation of En
Despite all signs of viability that the En double mutant mDA neurons showed
during the first 24 hours in culture (Fig.
2), it was possible that the cells were already committed to cell
death when the tissue was dissociated. Thus, the selective loss of mutant mDA
neurons during the cell mixing experiments
(Fig. 3) might only reflect
this commitment. To address this possibility, we silenced the En expression in
mDA neurons by RNA interference (RNAi). Recent experiments have shown that the
application of small interfering RNA duplexes (siRNA), 21-22 nucleotides in
length, leads to sequence-specific mRNA degradation in mammalian cell lines
(Elbashir et al., 2001a). We
performed all RNAi experiments in primary cell culture of ventral midbrain
derived from homozygous null mutants for the En2
(En2/), in order to increase the silencing
efficiency. This strategy was possible, as rescue experiments have shown that
En2 can functionally replace En1
(Hanks et al., 1995
) and
En2/ mutants show no phenotype with respect
to the mDA neurons (Simon et al.,
2001
). After isolation and dissociation of the E12 ventral
midbrain, cells were left growing in vitro for 72-96 hours and then
transfected with siRNA duplexes. At this point, mDA neurons exhibited an
elaborate network of neurites and they all expressed En1. Ninety-six
hours after transfection with two different En1-specific RNA
duplexes, the number of mDA neurons was reduced by about 25% compared with the
mock-transfected control cultures (Fig.
4A). Furthermore, we analyzed the siEn1 transfected
cultures at successive post-transfection time points
(Fig. 4B). The first
En1-negative DA neurons were detectable 12 hours after transfection, but the
numbers of TH-positive cells remained unchanged. We saw the first loss of DA
neurons at around 24 hours. The proportion of En1-negative cells increased
further, reaching a peak at around 48 hours post-transfection. The number of
TH-positive cells gradually declined until 96 hours and stabilised at around
75%. At this stage, En1-negative cells were rarely seen
(Fig. 4B). The mode of cell
death appeared to be the same as in the En double mutant embryos. Dying
En1-negative mDA neurons had rounded cell bodies containing condensed,
fragmented nuclei and showed a complete retraction of their neurites and the
presence of activated caspase 3 (Fig.
4C-J). The amount of cell loss after 96 hours, when no further
cell death was observed, suggested a transfection efficiency of 25%; however,
the proportion of En1-negative cells was never higher than 13% at any time
point of the experiment. There are two possible explanations for this
phenomenon: (1) the turnover rate of the En protein, or the onset of RNA
degradation, may be different in individual cells; or (2) suboptimal
silencing, where En1 is still detectable by immunohistochemistry, is
sufficient to induce cell death. The later possibility is unlikely, because we
never observed an apoptotic cell that was En1 positive.
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Discussion |
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With the exception of the apoptotic cell death, the most pronounced feature
of mDA neurons, which are deficient of the En genes, is the lack of axonal
outgrowth. This let us to speculate that the En genes cell-autonomously
regulate this process. Previous studies had shown that axonal pathway finding
is interrupted in a subpopulation of interneurons if En1 is not
present (Saueressig et al.,
1999). In the case of mDA neurons, the in vivo phenotype of the En
double mutants (Fig. 1A)
suggested a general inability of the mutant neurons to extend processes.
However, this is unlikely, as En double mutant mDA neurons from E12 embryos
grow out neurites for the first 24 hours, if dissociated and placed into
culture. Length and number of these neurites are indistinguishable from
littermate control cells. More likely, the deficit in the embryonic
environment, deletion of the neural tube rostral and caudal to the isthmus,
underlies the inability of the mDA neurons to extend an axon in vivo. However,
there is an alternative explanation for this observation. The dissociation by
enzymatic digestion may cause the reorganisation of cell surface proteins
leading to a delay in cell death. Axonal outgrowth, which is mostly dependent
on the assembly of intracellular cytoskeletal components, is probably not
affected by the digestion. Therefore under in vitro conditions, axonal
outgrowth is initiated and continues for the first 24 hours until apoptosis
sets in. Thus, the failing of axonal outgrowth in vivo may be just a
manifestation of the imminent programmed cell death and not related to
deficits in the environment of mDA neurons. This notion also fits the time
course of the cell death after silencing of En1 by the application of
siRNA duplexes (Fig. 4A). A way
to test this hypothesis would be the application of inhibitors of RNA
transcription or protein translation when the mutant cells are placed into
culture or when the siRNA duplexes are administered.
All experiments were performed in an En2/
background in order to increase the number of mutant embryos per litter and to
restrict the silencing by RNAi to just one gene, which was essential because
the transfection efficiency, if measured by the rate of induced cell death,
was 25%. For this reason, we cannot directly conclude that En2 is also
required for the prevention of apoptosis in mDA neurons. However, there is
strong evidence that this must be the case. In homozygote mutants null for
En1 or En2, alterations in the midbrain dopaminergic system
are minimal. The complete loss of mDA neurons only occurs when all four
alleles are deleted, strongly suggesting the two genes are functionally
interchangeable. The same can be said for the development of the entire CNS.
The replacement of En1 by En2 leads to the rescue of the
En1 phenotype with virtually no brain defect
(Hanks et al., 1995
). However,
the two genes are not completely identical. The limb abnormalities of
En1 mutants are neither suppressed, placing En2 into the En1
locus, nor in a different genetic background
(Bilovocky et al., 2003
).
Furthermore,
En1+/;En2/ mutant
mice are viable and fertile and there is no defect in the midbrain
dopaminergic system observable at P0. This is in contrast to the opposite
genotype,
En1/;En2+/; here,
the cluster of mDA neurons is reduced to a small domain
(Simon et al., 2001
). Thus,
with respect to SN and VTA one En1 allele is sufficient to produce a
P0 wild-type phenotype, but one En2 allele is not.
Several null mutations of transcription factors lead to the prenatal loss
of mDA neurons. In homologous recombinant mutant mice for Nurr1
(Nr4a2Mouse Genome Informatics)
(Wang et al., 2003;
Zetterström et al.,
1997
), mDA neurons fail to express their neurotransmitter
phenotype and begin to disappear at E15
(Wallen et al., 1999
). The
aphakia mice, which have a spontaneous null mutation of
Pitx3, (Semina et al.,
2000
) exhibit a specific loss of nigral DA neurons
(Nunes et al., 2003
;
Smidt et al., 2004
;
Van den Munckhof et al.,
2003
). In Lmx1b null mutants, the entire population of
mDA neurons is lost by E17 (Smidt et al.,
2000
). Furthermore, the null mutation for the trophic factor
TGF
(Blum, 1998
) leads
to a reduction of DA neurons in the SNc. The differential time courses and the
different degrees of neuronal loss suggest that the molecular bases for the
reduction in each of the mutant strains are probably unrelated. In this
context, the En double mutant phenotype is of particular interest as the mDA
neurons disappear the earliest and the entire population of mDA neurons is
affected, suggesting that the En genes exert fundamental control over a
mechanism that assures the survival of these cells.
Apoptosis is the mechanism leading to the loss of mDA neurons in many
genetic and experimental models of PD. Apoptotic profiles in the ventral
midbrain were observed in null mutants for Nurr1
(Saucedo-Cardenas et al.,
1998; Wallen et al.,
1999
) and in aphakia mice
(Van den Munckhof et al.,
2003
). Apoptotic cell death is induced in nigral dopaminergic
neurons by axotomy of the median forebrain bundle
(El-Khodor and Burke, 2002
),
striatal excitotoxic injury (Macaya et
al., 1994
) or by treatment with specific neurotoxins such as MPTP
(Tatton and Kish, 1997
) and
6-hydroxydopamine (He et al.,
2000
). Additionally, apoptosis plays a role in the regulation of
the numbers of mDA neurons during the first 14 days after birth
(Chun et al., 2002
;
Jackson-Lewis et al., 2000
),
probably reflecting a dependency of neurons on GDNF during this period
(Burke et al., 1998
;
Granholm et al., 2000
).
Apoptosis in mDA neurons is triggered in so many different experimental
paradigms, during normal development and during the pathological degeneration
of mDA neurons, it suggests that a common molecular pathway may be the cause.
Our RNA interference experiments showed that the activation of caspase 3, and
consequent cell death by apoptosis, occurs in some cells within 24 hours after
the application of the RNA duplex and silencing of En1. This is a
similar time scale as seen in 6-hydroxydopamine- and MPTP-induced degeneration
of the nigrostriatal DA system (Jeon et
al., 1995
; Sundstrom et al.,
1988
; Zuch et al.,
2000
) or the induction of cell death in sympathetic neurons after
withdrawal of NGF (Deckwerth and Johnson,
1993
) or activation of the low-affinity NGF receptor, p75
(Freidin, 2001
). Each of these
experimental models leads to the induction of apoptosis via the mitochondrial
(intrinsic) pathway (Vila and Przedborski,
2003
). The rapid induction of apoptosis, when En1 is silenced in
the mDA neurons by RNAi, make it plausible that the intrinsic pathway is also
triggered.
The total loss of mDA neurons in En double mutant mice, as early as E14,
and the speed with which apoptosis is induced in mDA neurons after silencing
of En expression, suggests that an essential molecular mechanism is affected.
It is possible that the degeneration of neurons seen in individuals with PD
and the loss of cells in the En double mutants have a common molecular origin.
In the En double mutant, large alterations in the level of gene expression
downstream of En1 and En2 are probably the reason for the
death of mDA neurons. During PD, the difference between pathological and
healthy levels of gene expression is probably small. This may be the reason
why most cases of PD cannot be traced back to a genetic mutation, despite the
fact that twin studies suggest a substantial genetic contribution
(Piccini et al., 1999).
Inherently, human mutant studies are less successful if they try to identify
small alteration in regulatory elements of a given gene. Recently, two
mono-allelic point mutations 5' to the coding region of Nurr1
have been shown to be associated with PD. The mutation leads to a reduction of
Nurr1 expression that, in turn, seems to affect the level of TH
expression (Le et al., 2003
).
In light of these studies, it is possible that a minor alteration in the
expression level of one of the En genes or small changes in the promoter
region of a downstream gene, which they regulate, leads to the slow
degeneration of nigral DA neurons in PD.
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ACKNOWLEDGMENTS |
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