Full text of DOI:10.1007/s004410000217

Cell Tissue Res (2000) 301:71–84
Digital Object Identifier (DOI) 10.1007/s004410000217
R E V I E W
Michael Sendtner · Geng Pei · Marcus Beck
Ulrich Schweizer · Stefan Wiese
Developmental motoneuron cell death and neurotrophic factors
Received: 6 March 2000 / Accepted: 14 March 2000 / Published online: 31 May 2000
©
Springer-Verlag 2000
Abstract During the development of higher vertebrates,
motoneurons are generated in excess. In the lumbar spi-
nal cord of the developing rat, about 6000 motoneurons
Changing signals involved in regulation of motoneuron
survival during embryonic and postnatal development
are present at embryonic day 14. These neurons grow out Experiments by Viktor Hamburger and others during the
axons which make contact with their target tissue, the first part of the twentieth century have shown that the
skeletal muscle, and about 50% of the motoneurons are developmental cell death of motoneurons is guided by
lost during a critical period from embryonic day 14 until influences provided by the target tissue (Hamburger
postnatal day 3. This process, which is called physiologi- 1934, 1958). Removal of limb buds in developing chick
cal motoneuron cell death, has been the focus of research embryos massively enhances the extent of motoneuron
aiming to identify neurotrophic factors which regulate cell death, and transplantation of an additional limb re-
motoneuron survival during this developmental period. duces the number of dying motoneurons. Thus, the indi-
Motoneuron cell death can also be observed in vitro vidual organism can react to deviations from genetically
when the motoneurons are isolated from the embryonic determined developmental programs, and this kind of
avian or rodent spinal cord. These isolated motoneurons plasticity might have contributed to the generation of a
and other types of primary neurons have been a useful highly complex nervous system in higher vertebrates
tool for studying basic mechanisms underlying neuronal during evolution. On the other hand, this high degree of
degeneration during development and under pathophysi- complexity also offers possibilities for disturbances such
ological conditions in neurodegenerative disorders. as gene mutations and dysregulation of gene expression
Accumulating evidence from such studies suggests that which then could be a cause of disease.
some specific requirements of motoneurons for survival
The idea that skeletal muscle plays a major role in
and proper function may change during development. regulating motoneuron survival during development has
The focus of this review is a synopsis of recent data on been recently challenged by the observation that mice
such specific mechanisms.
deficient for erb-B3, the receptor for glial growth factor
GGF), show a severe deficit of developing Schwann
(
Key words Motoneuron cell death · Neurotrophic
factors · Lumbar spinal cord · Rat
cells, and then, as a consequence, a significant reduction
(79%) in motoneurons (Riethmacher et al. 1997). These
data indicate that developing Schwann cells are at least
as important for motoneurons as skeletal muscle. How-
ever, these data could indicate that motoneurons first be-
come dependent on muscle-derived neurotrophic support
and then, subsequently, on Schwann-cell-derived factors.
Indeed, when skeletal muscle is destroyed by a cre/loxP-
Work in the author’s laboratory is supported by the Deutsche mediated strategy (Grieshammer et al. 1998), motoneu-
Forschungsgemeinschaft, Grants To61/8-4, SFB 465TPA3 and
ron survival in the lumbar spinal cord is highly dimin-
ished during embryonic development, as expected from
BMBF 01KO9705
M. Sendtner ( ) · G. Pei · M. Beck · U. Schweizer · S. Wiese
the early data obtained from experiments in chicks when
limb buds are removed (Oppenheim 1985). In conclu-
sion, it has to be assumed that neurotrophic factors from
various sources function together in regulating survival
and functional integrity of motoneurons during both
embryonic and postnatal development.
✉
Klinische Forschergruppe Neuroregeneration,
Neurologische Universitätsklinik Würzburg,
Josef-Schneider-Str. 11,
9
7080 Würzburg, Germany
e-mail: sendtner@mail.uni-wuerzburg.de
Tel.: +49 931 201 5767, Fax: +49 931 201 2697

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2
This scenario appears even more complex with otherwise inducible manner by use of the Cre/loxP and
respect to early findings that motoneuron cell death in other recombination techniques will help to understand
vertebrates can also occur in a target-independent man- such processes and thus could provide a basis for ratio-
ner. Rita Levi-Montalcini and others have shown (Levi- nal design of therapeutic strategies for treatment of vari-
Montalcini 1950; O’Connor and Wyttenbach 1974; ous forms of motoneuron disease.
Oppenheim et al. 1989) that, during early development
of the chick embryo around day 4, massive cell death
can be observed in a specific population of motoneurons Neurotrophic factors for motoneurons
in the ventral cervical spinal cord. This type of cell death
is highly synchronized, in contrast to motoneuron cell Since the discovery of nerve growth factor (NGF) as a
death observed in this and other regions of the develop- prototypic target-derived neurotrophic molecule that reg-
ing chick spinal cord between embryonic days 6 and 10, ulates survival of subpopulations of sensory paraverte-
which roughly corresponds to embryonic day 14 to post- bral sympathetic neurons, more than a dozen neurotro-
natal day 3 in the mouse or rat. A recent study (Yaginuma phic factors have been identified and characterized on a
et al. 1996) has shown that this type of early motoneuron molecular level (Table 1 ). This list includes factors
cell death occurs in a target-independent manner and may called neurotrophins, which are related to NGF (Barde
be determined by a cell-autonomous program.
1990). At least three neurotrophins, brain-derived neuro-
Taken together, these data suggest that cell death pro- trophic factor, neurotrophin-4 and neurotrophin-3, but
grams in motoneurons probably change during develop- not NGF, support motoneuron survival (Sendtner et al.
ment, and at least some of the motoneurons pass through 1992a; Hughes et al. 1993; Henderson et al. 1993).
an early phase when cell-autonomous and/or target- Motoneurons are also supported by other families of
independent processes regulate cell death and then neurotrophic molecules. Among these are factors which
undergo a phase when signals from skeletal muscle play exert their survival-promoting activity through complex
a major role. Subsequently, signals from Schwann cells membrane receptors involving gp130 and LIFR-β
such as CNTF and other survival factors contribute to (Nakashima and Taga 1998). Factors which bind to such
the maintenance of motoneurons, and arising evidence complexes are ciliary neurotrophic factor (Arakawa et al.
from experiments with various mouse mutants suggests 1990), leukemia-inhibitory factor (Hughes et al. 1993),
that postnatal survival of motoneurons becomes increas- cardiotrophin-1 (Pennica et al. 1995) and the recently
ingly dependent again on cell autonomous processes identified cardiotrophin-like cytokine (CLC) (Pennica
such as sufficient expression of the “survival of moto- et al. 1995; Senaldi et al. 1999; Shi et al. 1999).
neuron” (SMN) protein (Jablonka et al. 2000; Monani et
Motoneuron survival in culture is also supported by
al. 2000) and protection from glutamate-mediated toxicity. members of the glial-derived neurotrophic factor
Further research needs to show how these signals and the (GDNF) gene family of transforming growth factor-β-
intracellular signaling pathways function together and related survival factors (Henderson et al. 1994; Zurn et
how the motoneurons react to disturbances of individual al. 1994). Factors which support motoneuron survival
signals. The availability of mouse mutants in which indi- include GDNF (Henderson et al. 1994), neurturin (Klein
vidual signals are disrupted either by classical homolo- et al. 1997), persephin (Milbrandt et al. 1998), and
gous recombination or in a cell-type-specific manner or artemin (Baloh et al. 1998). These molecules act through
Table 1 Neurotrophic factors
Receptor on motoneurons
for motoneurons and their
receptors
1
. Neurotrophins
NTR
Brain-derived neurotrophic factor (BDNF)
Neurotrophin-3 (NT-3)
Neurotrophin-4/5 (NT-4/5)
p75 , trk-B
NTR
p75 , trk-C
NTR
p75 , trk-B
2
. CNTF/LIF family
Ciliary neurotrophic factor (CNTF)
Leukemia inhibitory factor (LIF)
Cardiotrophin-1 (CT-1)
CNTFRα, LIFRβ, gp130
LIFRβ, gp130
?, LIFRβ, gp130
?, LIFRβ, gp130
Cardiotrophin-1-like cytokine (CLC)
3
4
. Hepatocyte growth factor/scatter factor (HGF/SF)
c-met
. Insulin-like growth factors
IGF-I
IGF-II
IGFR-1
IGFR-1, mannose-6P receptor
5
. Glial-derived neurotrophic factor and related factors
Glial-derived neurotrophic factor (GDNF)
GFRα1, c-ret
GFRα2, c-ret
GFRα4, c-ret
GFRα3, c-ret
Neurturin (NTR)
Persephin
Artemin

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3
receptors involving the c-ret tyrosine kinase, and specific Moreover, it is very likely that these factors influence
α-receptors (Baloh et al. 1997). specific properties of motoneurons such as the regulation
Other factors with survival-promoting activity for of transmitter synthesis and release, axon outgrowth and
motoneurons include insulin-like growth factor (IGF)-1 dendrite arborization as well as synaptic stability (re-
and IGF-2 (Arakawa et al. 1990; Neff et al. 1993) and viewed in Sendtner 1998). It is therefore not surprising
hepatocyte growth factor (HGF) (Wong et al. 1997; that individual factors have not been identified so far that
Ebens et al. 1996; Yamamoto et al. 1997). In cultures of serve prototypic functions, i.e., the control of survival
isolated embryonic chick spinal motoneurons, the sur- during the period of physiological motoneuron cell
vival-promoting effects of IGFs are relatively low, in death.
contrast to cultures of embryonic rat motoneurons, which
can be maintained in serum-free medium (Hughes et al.
1
993). However, when IGFs are combined with other Cellular signaling pathways responsible
neurotrophic factors such as CNTF, this leads to supra- for neurotrophic-factor-mediated survival
additive survival effects (Arakawa et al. 1990). This was of motoneurons: receptors for neurotrophic factors
one of the first demonstrations that neurotrophic factors as key regulators
potentiate each other in supporting motoneuron survival,
suggesting that survival of motoneurons in vivo depends Recent years have seen significant progress in the under-
on a complex cooperation of more than one factor. In standing of signaling pathways which mediate the vari-
other words, a single motoneuron may depend on more ous functions of neurotrophic factors in neuronal cells.
than one neurotrophic factor, and development, survival The neurotrophins have served as a prototypic gene fam-
and proper function of these cells could be regulated by ily for investigation of the signaling pathways which
such factors in a way that they have to function together regulate survival and other effects in primary neurons
in a well-balanced manner.
(Segal and Greenberg 1996). A significant contribution
HGF is a heterodimeric protein with similarities to to this research was provided by investigations employ-
plasminogen. However, it lacks enzymatic activity ing the rat pheochromocytoma cell line PC-12. Signaling
(
Weidner et al. 1991). Interestingly, only lumbar moto- through high-affinity neurotrophin receptors, which in-
neurons from 5-day-old chick embryos survive with clude members of the trk gene family, involves several
HGF, but not motoneurons from thoracic or cervical spi- pathways. Initiation of this signaling depends on the cy-
nal cord (Novak et al. 2000). The c-met tyrosine kinase tosolic adapter protein Shc, the activation of PLC-γ and
is expressed in lumbar but not in thoracic motoneurons the activation of the PI-3 kinase/AKT pathways directly
between embryonic days 5 and 10 during the period of at the level of the trk transmembrane tyrosine kinases.
physiological motoneuron cell death in chick. However,
The Shc adapter proteins have been the focus of re-
the expression of c-met in lumbar motoneurons seems to search, as they can couple trk-signaling to the Ras/MAP-
be regulated by other target tissue-derived factors than kinase pathway, which has been shown to be involved in
HGF. This conclusion is based on the observation that promoting neuronal survival and neurite outgrowth. Ex-
the massive cell death of motoneurons which occurs in periments with PC-12 cells (Bar-Sagi and Feramisco
the lumbar spinal cord after limb bud removal cannot be 1985) and with primary sympathetic neurons (Borasio et
reduced by treatment with HGF, probably because the al. 1993) have shown that activated Ras can support dif-
c-met receptor is downregulated by limb-bud removal ferentiation and survival of neuronal cells. At least in
and signals which lead to expression of this receptor are PC-12 cells, sustained activation of the MAPK pathway
thus removed. Thus, HGF is another example of a neuro- (Traverse et al. 1992; Wixler et al. 1996; Aletta 1994)
trophic factor which influences survival only of subpop- seems to be responsible for the differentiation versus mi-
ulations of motoneurons, and needs cooperation with togenic effects of NGF in comparison to epidermal
other signals in order to exert its survival-promoting growth factor (EGF), and it has been suggested that sus-
effect under physiological conditions. The list of mole- tained activation of the MAPK pathway is also responsi-
cules that support motoneuron survival is still growing, ble for the neurotrophin-mediated survival effect in neu-
and it is expected that it will become even longer during ronal cell lines and primary neurons. Recently, a second
the next few years.
Ras-independent pathway for activation of the MAPK
Despite this richness of identified molecules, very pathway has been shown to be involved in mediating the
little is known about the role they play during embryonic differentiation effects of NGF (York et al. 1998). This re-
development. The high number of neurotrophic factors directs the focus of interest on activation mechanisms
which can act on motoneurons indicates that regulation upstream from Ras and other small GTP-binding pro-
of motoneuron survival and function is a highly complex teins which transduce the signal from receptor molecules
process which is influenced by an as yet only partially to the MAPK and other intracellular signaling pathways.
characterized cooperation of these molecules. In addi-
In neurons, several forms of Shc exist, which are
tion, some of these molecules are only expressed postna- named ShcA, ShcB, and ShcC/N-shc (Cattaneo and
tally, indicating that the requirement of motoneurons for Pelicci 1998). The expression of these Shc isoforms is
these factors changes during development, and that post- under strict control during development, and only little is
natal survival is also controlled by neurotrophic factors. known so far as to whether differences in the expression

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4
of these Shc isoforms influence signal transduction tion of the NFκB pathway (Ozes et al. 1999; Romashkova
downstream of trk receptors, for example by determining and Makarov 1999). It remains to be seen which of these
downstream targets involved in mediating cellular ef- downstream pathways is responsible for motoneuron sur-
fects of neurotrophic factors including survival of specif- vival during various stages of development and in the
ic neuronal cell types at a specific stage of development. adult.
The ratio of ShcA and ShcC /N-Shc expression deserves
A third important pathway which could be of impor-
particular attention. ShcC/N-Shc has been detected as a tance in particular for motoneuron survival is the activa-
neuron-specific Shc isoform (Nakamura et al. 1996) tion of PLCγ1 (Obermeier et al. 1994; Tinhofer et al.
which mediates the coupling of trk signaling to Ras acti- 1996; Segal and Greenberg 1996). Activation of this
vation. Whereas ShcA is expressed in a widespread man- pathway leads to increased release of free Ca2 from in-
ner in virtually all types of cells, ShcC expression is only tracellular stores, which in turn can activate cyclic AMP
found in the nervous system. Interestingly, the upregula- response-element-binding protein (CREB) (Finkbeiner et
+
2
+
tion of ShcC corresponds to a downregulation of ShcA al. 1997). In addition, elevated intracellular Ca levels
expression in the brain at a developmental stage when have also been described to activate the small GTP-bind-
neuronal cells become postmitotic (Conti et al. 1997) ing protein Rap1 but not Ras (Grewal et al. 2000). This
and at least some populations of neurons including the leads to activation of B-Raf but not to activation of
motoneurons become dependent on neurotrophic factors Raf-1. In developing motoneurons, B-Raf is expressed at
for their survival. It is tempting to speculate that this relatively high levels during the period of physiological
switch from ShcA to ShcC expression might be responsi- cell death (G. Pei and M. Sendtner, unpublished observa-
ble for a switch in response to neurotrophic factors from tions). Thus a link between various pathways could ex-
a differentiation signal to a survival signal (Segal and ist, and increased levels of Ca2 could contribute to acti-
Greenberg 1996). This could be of importance for under- vation of Rafs, which seem to play a central role both in
standing how motoneurons become responsible to neuro- activation of the MAPK pathway and also as effector
+
trophic factors for their survival during development.
kinases for bcl-2 which phosphorylate BAD and thus in-
Recent evidence from mice in which the Shc-binding hibit its proapoptotic activity (Wang et al. 1994, 1996).
site in trk-B was mutated (Minichiello et al. 1998) sug-
gests that differences between various neurotrophic fac-
tors exist in the utilization of this pathway for mediating The role of afferent activity and extracellular matrix
survival and neurite outgrowth effects. Mice in which in the survival of motoneurons
the Shc-binding site of trk-B is mutated show that NT-4-
mediated survival effects were more dramatically re- In various types of neurons, removal of afferent input
duced than BDNF-dependent differentiation or survival. leads to increased cell death in vivo (Okado and Oppen-
These mice display a complete loss of the NT-4-depen- heim 1984). This finding could correspond to observa-
dent D-hair sensory cells but no change in BDNF-depen- tions that cultured neurons can be maintained under con-
dent slowly adapting mechanotransducing sensory neu- ditions which mimic depolarization such as elevated po-
rons. However, survival of motoneurons has not been in- tassium concentration (Wakade et al. 1983) or even addi-
vestigated in such mice so far, and it remains to be dem- tion of glutamate receptor agonists at optimal concentra-
onstrated whether survival or functional properties in tions (Meyer-Franke et al. 1995). However, various types
subpopulations of motoneurons are altered when the of neurons differ in their capacity to survive in culture in
Shc-binding site is mutated in the trkB receptor.
the presence of increased levels of potassium or gluta-
+
Nevertheless, this report showed that significant dif- mate. Whereas 35 mM K in the culture medium can
ferences exist between various ligands in the activation support survival of sympathetic neurons derived from
of downstream pathways, even if shared receptor compo- 10-day-old chick embryos (Wakade et al. 1983), moto-
nents such as trkB in the case of BDNF and NT-4 are neurons from 5- or 6-day-old chick embryos cannot sur-
involved.
vive for periods longer than 3 days under the same cul-
Besides the Ras/MAPK pathway, a second pathway ture conditions. In this respect, motoneurons show simi-
involving activation of PI-3 K seems to be highly impor- lar behavior to retinal ganglion cells, which also do not
tant for neuronal survival (Dudek et al. 1997). Direct survive when the potassium concentration in the culture
association of PI-3 K with trk receptors has been found medium is elevated (Meyer-Franke et al. 1995). On the
under specific experimental conditions (Obermeier et al. other hand, survival with the neurotrophic factor BDNF
1
993). However, it is now widely accepted that Shc sig- was strongly enhanced in isolated retinal ganglion cells
naling plays a major role in the activation of PI-3 K when the cells were depolarized or even when N-methyl-
downstream of trks in neuronal cells (Baxter et al. 1995; D-aspartic acid (NMDA) or kainic acid was given to
Greene and Kaplan 1995; Datta et al. 1997). The PI-3 ki- these cultures. The effect could be mediated by elevation
nase in turn leads to activation of Akt, a serine/threonine of cAMP, as inhibitors of protein kinase A could inhibit
kinase with a broad spectrum of substrates including the the potentiation of the BDNF survival effect by in-
proapoptotic Bad (Datta et al. 1997), caspase-9 (Cardone creased potassium or glutamate agonists in the culture
et al. 1998), the forkhead transcription factor FKHRL1 medium. These data also suggest that intracellular Ca2
+
(
Brunet et al. 1999) and IKKα kinase, leading to activa- elevation is responsible for this effect.

7
5
Glutamate toxicity is considered a major pathomecha- (Meyer-Franke et al. 1998). This could be of major phys-
nism in amyotrophic lateral sclerosis (ALS) (Choi 1992), iological relevance, as many neurotransmitters act via G-
the most abundant form of motoneuron disease in adult protein-coupled receptors, and thus changes in cAMP
humans. Indeed, an overwhelming amount of indirect levels in motoneurons might occur by specific signaling
evidence for this hypothesis has been found, including from other types of cells. In the absence of neurotrophic
increased glutamate content in the CSF of ALS patients factors, cAMP only promotes short-term survival of spi-
(
Rothstein et al. 1992, 1996), and splicing defects in glu- nal motoneurons in culture when merosin is used as a
tamate transporter mRNAs in the brain of ALS patients substrate. However, under these specific conditions, the
Aoki et al. 1998), which are thought to cause chronically combination of an elevation of intracellular cAMP and
(
enhanced synaptic glutamate levels. However, direct neurotrophic factors leads to prolonged survival of moto-
proof that enhanced glutamate causes motoneuron de- neurons over periods exceeding 3 weeks. Similarly, ele-
generation in human motoneuron disease is hard to ob- vation of cAMP levels has also been shown to increase
tain. For example, clinical studies with specific gluta- survival of superior cervical ganglion neuron and cere-
mate receptor inhibitors have not been successful so far bellar granule cells. These cAMP-mediated survival
(
Gredal et al. 1997), and any evidence as to whether they effects seem to be independent of PI-3 K activation,
can block the progression of the disease is still lacking. since inhibitors such as Wortmannin could not abolish
Although riluzole, a drug with proven efficacy for treat- the pro-survival effects of cAMP. A first indication of
ment of ALS (Bensimon et al. 1994), is thought to inter- how these survival pathways could be linked was given
fere with glutamatergic neurotransmission, it is not clear by the demonstration that cAMP elevation can increase
whether the effect occurs at the presynaptic or post- the recruitment of the trk-B receptor to the plasma mem-
synaptic site, or even by interference with other patho- branes in retinal ganglion cells (Meyer-Franke et al.
genic mechanisms than glutamatergic signaling such as 1998). This mechanism could be of considerable rele-
presynaptic potassium channels.
vance for the development of therapeutic strategies im-
Embryonic motoneurons express NMDA receptors plying neurotrophic factors. Studies with primary neu-
Kalb et al. 1992) and α-amino-3-hydroxy-5-methyl-4- rons in cell culture have shown that BDNF exposure
(
isoxazolepropionic acid (AMPA) receptors including leads to a rapid downregulation of the cell membrane ex-
subtypes of AMPA receptors which mediate Ca2 influx posure of trk-B (Carter et al. 1995), and therapeutic ad-
in response to glutamate (Metzger et al. 2000). When ministration of BDNF at high concentrations to axotomi-
these motoneurons are exposed to enhanced concentra- zed motoneurons after facial nerve transection only leads
tions of glutamate or NMDA in vitro, motoneuron cell to transient effects (Vejsada et al. 1995), even if BDNF is
death is not enhanced (Metzger et al. 1998). This seems administered chronically with osmotic pumps or repeat-
surprising with respect to the observation that massive edly by systemic injections. This indicates that inhibition
cell death occurs under the same conditions, for example of transfer of trk-B from intracellular to cell membrane
in cultures of cortical neurons (M. Sendtner, unpublished compartments could play a role in the downregulation of
observations). Even when the motoneurons are depolar- trk-B survival signals under physiological conditions.
+
2
+
ized in order to remove the Mg block of NMDA recep- However, it is likely that additional cellular mechanisms
tors, this does not influence the survival of the motoneu- contribute to the specific effects of cAMP on motoneu-
rons in cell culture, suggesting that isolated embryonic ron survival.
motoneurons are quite resistant to glutamate toxicity. On
These data suggest that neurotrophic factors should
the other hand, NMDA or glutamate exposure does not not be considered as the only players which regulate mo-
potentiate the survival effect of BDNF or other neurotro- toneuron cell death under physiological conditions. In-
phic factors, as observed with isolated retinal ganglion deed, motoneuron survival in culture in the presence of
cells (Meyer-Franke et al. 1995). Despite this negative neurotrophic factors strongly depends on the presence of
effect, motoneurons show specific responses to enhanced components of the extracellular matrix. Laminin was one
glutamate levels in the culture medium. Glutamate treat- of the first molecules which was found to potentiate the
ment leads to specific inhibition of dendrite growth in a survival effect of neurotrophic factors (Edgar et al.
fully reversible manner. Axon outgrowth was not affect- 1984). When motoneurons from 6-day-old chick embry-
ed under the same culture conditions by glutamate expo- os are isolated and cultured in the presence of laminin,
sure, suggesting that glutamate could play an important about 55% of these isolated motoneurons can be main-
role in plasticity of dendrite growth and synapse forma- tained for periods longer than 1 week (Arakawa et al.
tion between motoneurons and other types of neurons 1990) with CNTF. However, this survival effect signifi-
within the spinal cord.
cantly drops when the concentration of laminin used to
coat the culture dishes is reduced from 10 to 1 µg/ml
(
Table 2 ). In the absence of laminin, the survival effect
Modulatory role of cAMP for motoneuron survival
of CNTF even drops to 3.7%, which is not statistically
different from control when motoneurons are cultured in
Recent studies have demonstrated that elevation of the absence of neurotrophic factors (2.4% survival).
cyclic AMP (cAMP) can provide a survival signal for These data indicate that integrin signaling cooperates
motoneurons even in the absence of neurotrophic factors with neurotrophic factors in mediating motoneuron sur-

7
6
Table 2 Influence of laminin on CNTF-mediated survival of iso- embryos (Raoul et al. 1999). Interestingly, Fas and Fas
lated embryonic chick motoneurons (reproduced with permission
from Sendtner et al. 1991)
ligand are expressed early during development in moto-
neurons, and it has been suggested that the coexpression
of these two molecules in principle allows cell-autono-
mous cell death programs. When embryonic motoneu-
rons were cultured for 3 days with neurotrophic factors,
they became resistant to Fas-induced cell death, either by
induction of interference with downstream effectors such
as the activated caspase-8 and caspase-3, or by allowing
maturation of motoneurons to a stage when they are
resistant to cell-autonomous cell death programs, or by
both mechanisms. Thus, Fas and Fas ligand could play a
role during early phases of motoneuron cell death when
these cells do not yet depend or even cannot react to neu-
rotrophic factors, and further studies, for example in-
volving Bid-deficient mice which are resistant to Fas-
induced cell death, could be of great help in understand-
ing the physiological role of Fas-mediated cell death for
motoneurons.
Neurotrophic
factor
Amount of laminin Survival of motoneurons
used for culture
dish coating (µg)
after 3 days in culture
(% of originally plated cells)
CNTF
FGF-2
10
10
55.4
44.4
95.8
2.4
22.5
22.4
53.3
0.0
3.7
8.9
12.5
0.0
CNTF+FGF-2 10
None
10
1
CNTF
FGF-2
1
CNTF+FGF-2
None
1
1
CNTF
FGF-2
CNTF+FGF-2
None
–
–
–
–
The p75 neurotrophin receptor (p75NTR) shares struc-
vival, and it will be interesting to investigate whether in-
tegrin signaling influences cAMP levels in motoneurons tural and functional similarities with other transmem-
and thus functions together with neurotrophic factors in brane molecules of the Fas/Apo-1/CD95 and tumor
regulating specific functions in motoneurons.
necrosis factor receptor-1 family (see Nagata 1997 for
Indeed, the effect of elevated cAMP on motoneuron review). In a variety of cellular contexts in vitro and in
NTR
survival is strongest when the motoneurons are cultured vivo, p75
mediates cell death after binding of NGF,
on merosin (Meyer-Franke et al. 1998), a synapse-specific in particular when trkA is not expressed (Chao et al.
isoform of laminin. When motoneurons from 15-day-old 1998; Friedman and Greene 1999). Injection of neutral-
rat or 14-day-old mouse embryos are cultured on a lami- izing antibodies into the eye of early chick embryos has
NTR
nin substrate (S. Wiese and M. Sendtner, unpublished shown that NGF and p75
are involved in mediating
observations), the survival effect of elevated cAMP is early cell death of retinal ganglion cells which have just
much weaker. Thus, an important question for further re- been formed and are starting to grow axons (Frade et al.
search is whether merosin and laminin differentially in- 1996; Frade and Barde 1998a). In this respect, NGF and
2
+
NTR
fluence the rise of free Ca after cAMP stimulation. p75
are involved in an early phase of cell death
This may also be of importance for the elucidation of which is not regulated by the target tissue. In this specif-
other specific effects of neurotrophic factors beyond the ic case, the source of NGF was microglial cells which
support of survival, such as axon outgrowth, synaptic had invaded the retina during these early developmental
stability and fine-tuning of synapse activity.
stages (Frade et al. 1996).
NTR
The role of p75
as a mediator of cell death has also
been shown in basal forebrain cholinergic neurons (Yeo
Target-dependent and target-independent cell death et al. 1997), and overexpression of the cytoplasmic part
NTR
NTR
of motoneurons: a role for Fas and p75 signaling of the p75
in mice (Majdan et al. 1997a) leads to sig-
in motoneurons?
nificant losses of several populations of neurons includ-
ing motoneurons in the facial nucleus. Thus, it appears
Genetic determination of cell death plays an important intriguing that p75NTR could have similar functions in
role in the development of lower organisms such as C. motoneurons, in particular when this receptor is ex-
elegans. The ces-2 gene apparently acts cell autono- pressed at high levels after axotomy. Indeed, when the
mously to trigger programmed cell death (Metzstein et expression of this receptor was abolished in adult
NTR
al. 1996; Ellis and Horvitz 1991). Ces-2 is expressed p75 –/– mice, survival and regeneration of axotomi-
specifically in two distinct pharyngeal motoneurons in zed motoneurons was improved in comparison to control
the worm and initiates death in these specific cells. Such animals (Ferri et al. 1998), and overexpression of the cy-
NTR
cell-autonomous cell death genes have not been identi- toplasmic part of p75
enhanced lesion-induced cell
fied so far in motoneurons from higher vertebrates, death of facial motoneurons in adult mice (Majdan et al.
although it is not unlikely that they could play a role, for 1997). Moreover, application of NGF to axotomized sci-
example during an early phase of development when atic (Miyata et al. 1986) or facial (Sendtner et al. 1992b)
motoneuron cell death apparently occurs in a target- motoneurons significantly increased the rate of cell death
independent manner. The cell death receptor Fas and the in rats or mice (Wiese et al. 1999a). These results are
NTR
corresponding Fas ligand are expressed in early moto- compatible with the assumption that p75
is a cell
neurons, and Fas has been found to transmit a cell death death receptor in developing motoneurons. On the other
signal in cultured motoneurons from 12.5-day-old mouse hand and in contrast to other neuronal populations (Van

7
7
der Zee et al. 1996; Bamji et al. 1998; Yeo et al. 1997),
the number of motoneurons in 6- to 9-week-old
NTR
p75 –/– mice was shown to be reduced in comparison
to wild-type mice, as revealed by quantification of facial
motoneuron numbers and axon profiles in brain stem and
peripheral nerves (Ferri et al. 1998; Wiese et al. 1999a).
However, in the case of basal forebrain cholinergic neu-
rons, contradictory results have been obtained in the
meantime so that at least one of the originally published
reports seems to be not reproducible, and the findings
thus do not support a proapoptotic function through the
p75 neurotrophin receptor in postnatal basal forebrain
cholinergic neurons (Van der Zee et al. 1996; Bamji et al.
1
998; Yeo et al. 1997).
Motoneurons not only express high levels of p75NTR
after axotomy, but also under physiological conditions
during early development. Expression of this receptor
molecule apparently occurs before the motoneurons have
gained contact with skeletal muscle and before these
motoneurons become responsive at least to BDNF for
their survival. In order to test the possibility that NGF
NTR
and p75
are mediators of the early phase of motoneu-
ron cell death in brachial spinal cord, Frade and Barde
have investigated cell death in the spinal cord of
NTR
p75 –/– and NGF–/– mice between E10.5 and E12.5,
before the period of classical target-dependent cell death
starts (Frade and Barde 1999). In such animals, an in-
crease in the thickness of the mantle zone was observed.
However, the number of islet-1-positive motoneurons
was not increased in the ventral portion of the spinal
cord of these mutant mice.
NTR
In order to test the role of p75
and the actions of
NGF on developing motoneurons during the classical pe-
riod of target-dependent motoneuron cell death in more Fig. 1 NGF antagonizes BDNF-mediated survival of mouse
motoneurons in culture. Motoneurons were isolated from lumbar
detail, we have developed techniques for isolation of
spinal cord of 14-day-old mouse embryos (Wiese et al. 1999a).
motoneurons from lumbar spinal cord of 14-day-old
Survival of motoneurons was determined by counting surviving
NTR
wild-type and p75 –/– mice (Wiese et al. 1999a). NGF
motoneurons after 3 days in culture. Values shown are percentages
did not support long-term survival of isolated mouse of initially plated neurons in the culture dish. BDNF and NGF
were added at concentrations indicated at 10 or 100 pg/ml. The
motoneurons. On the contrary, it reduced the survival ef-
NTR
survival effect of BDNF is significantly reduced in the presence of
NGF at equimolar concentrations, and completely abolished when
NGF is given at doses 10 times higher than BDNF. Note that the
fects of BDNF and NT-3 in wild-type but not p75 –/–
motoneurons. On first inspection, these data favor the
NTR
model of p75
as a cell death receptor on motoneu- maximal concentration of NGF in this experiment was 100 pg/ml,
rons. However, the quantities of NGF which interfered which is below the concentration thought to saturate low-affinity
neurotrophin receptors. NGF could not reduce the survival re-
with the survival responses of BDNF and NT-3 were
sponse initiated by GDNF, even when the concentration was in-
–
13
very low (7×10 M) (Fig. 1). They did not reach the
creased to a level of 1000 pg/ml, thus suggesting that NGF acts as
a specific antagonist, probably at the receptor level, rather than
levels necessary for saturating those low-affinity binding
sites on primary neurons which are thought to corre- acting as an independent cell death effector on these cultured
NTR
motoneurons
spond to p75
receptors (Meakin and Shooter 1992;
Rodriguez-Tébar et al. 1990). Recently, it has been de-
NTR
scribed that p75
can be expressed in neuronal cells in survival at later developmental stages in chick (Qin-Wei
NTR
a conformation conferring high-affinity binding of NT-3 et al. 1994) when p75
Dechant et al. 1997). However, under the same experi- al. 1988) suggest that the presence of p75
mental conditions NGF did not bind to p75
added at 10 M.
expression is low (Ernfors et
NTR
(
could en-
when hance the specificity of BDNF and NT-3. Determination
of motoneuron cell numbers in newborn NGF–/– mice or
NTR
–
12
Observations that very low quantities of NGF inter- 1-week-old NGF+/– mice after facial nerve lesion at
fere with BDNF and NT-3 effects in neurons could be birth could help to further define whether endogenous
NTR
explained by a model in which the p75
is part of the NGF interferes at neurotrophin receptors in developing
high-affinity receptor for BDNF and probably also NT-3. and/or postnatal lesioned motoneurons. Such experi-
Previous observations that NGF enhances motoneuron ments are underway in several laboratories.

7
8
Reduction of NT-3- and BDNF-mediated survival by
NTR
NGF is not seen in p75 –/– motoneurons. This obser-
NTR
vation also suggests that p75
receptors are compo-
nents of the high-affinity binding sites for NT-3 and
BDNF on E14 lumbar motoneurons and do not constitute
separate NGF receptors. We have also observed that the
BDNF dose necessary for half-maximal survival is shift-
–
12
ed from 1.2×10
M in wild-type motoneurons to
–
12
NTR
5
.4×10
M in p75 –/– motoneurons. This supports
previous data that trk homodimers are not equivalent to
high-affinity neurotrophin receptors in primary neurons
(
Mahadeo et al. 1994; Wolf et al. 1995). Our data suggest
NTR
that p75
for BDNF and possibly also NT-3 on motoneurons. Thus
p75
trk-C on the cell surface of motoneurons, and only part if
is part of the high-affinity receptor complex
NTR
is expected to interact physically with trk-B and
NTR
any of the p75
transmembrane molecule receptors
should exist separately and function independently as cell
death receptors on these cells. In the latter case, one
NTR
would expect that NGF signaling through p75
should
also reduce the survival response mediated through
CNTF or GDNF receptors. However, even high quantities
of NGF (20 ng/ml) known to saturate low-affinity recep-
tors on neuronal cells (Rodriguez-Tébar et al. 1990) did
not reduce CNTF- and GDNF-mediated survival of moto-
neurons. This was surprising insofar as the CNTF-
mediated survival response of embryonic chick mesence-
phalic trigeminal neurons was reduced by NGF (Davey
and Davies 1998). We can exclude the possibility that
NTR
CNTF and GDNF downregulate p75
expression in
motoneurons, as motoneurons treated either with CNTF,
NTR
GDNF, or BDNF showed similar intensities of p75
Fig. 2a–c NGF and CNTF lead to a rapid upregulation of the avi-
immunostaining after 5 days in culture. Thus, we con-
in neuronal cells differs de- mammalian IAPs in cultured sensory and sympathetic neurons.
pending on cell type and developmental stage. Whether IAPs are potent inhibitors of caspases, and thus could participate
an inhibitor of T-cell apoptosis (ITA) protein, a homologue of
NTR
clude that the role of p75
in the survival response of neurons to neurotrophic factors. See
such differences are caused by the presence or absence of
Wiese et al. (1999b) for details (reproduced with permission from
NTR
downstream effectors of trk and p75
signaling, or sim-
this publication)
NTR
ply by the fact that apoptosis through p75
requires
higher expression levels than those found in motoneurons
from E14 mouse spinal cord, remains to be seen.
molecules are not physiologically necessary for moto-
neuron survival during development. Interestingly, moto-
neuron cell death in bcl-2–/– mice is enhanced during
Characterization of downstream signaling pathways the postnatal period, leading to loss of around 40% of fa-
which mediate the neuronal survival response
to neurotrophins and CNTF: the role of members
of the IAP family
cial motoneurons of 6-week-old mice. This suggests that
bcl-2 becomes important for postnatal survival at least of
subpopulations of motoneurons. However, this does not
mean that bcl-2-deficient motoneurons lose their respon-
Not very much is known about the downstream signals siveness to neurotrophic factors. Both BDNF and CNTF
involved in execution of motoneuron cell death cell and are still capable of rescuing motoneurons from lesion-
neurotrophic-factor-mediated survival. Research on the induced cell death after facial nerve transection in new-
role of bcl-2 has led the way to our present knowledge. born animals. Nevertheless, survival in response to these
Overexpression of bcl-2 can significantly reduce the neurotrophic factors was lower in bcl-2-deficient mice,
extent of motoneuron cell death during development indicating that subpopulations depend on bcl-2 for
(
Martinou et al. 1994; Dubois-Dauphin et al. 1994). survival after nerve lesion, and/or that compensation by
However, mice in which bcl-2 expression is abolished by other antiapoptotic members of the bcl-2 family is only
homologous recombination show only a small reduction incomplete in these cells.
of motoneuron numbers at birth (Michaelidis et al.
996), indicating either that bcl-2-related molecules can (Knudson et al. 1995; Deckwerth et al. 1998) are resis-
substitute for this deficiency or that bcl-2 and related tant to cell death after neurotrophic factor deprivation
Observations that neurons from BAX-deficient mice
1

7
9
Fig. 3 Signaling pathways involved in neurotrophic-factor-medi- the presence of NGF. Based on these observations, it was
ated survival of embryonic motoneurons. Recent studies by
speculated that NGF leads to a rapid production of an in-
Deshmukh and Johnson (1998) have shown that injection of cyto-
tracellular protein, which protects cells from the proapo-
chrome c into the cytoplasm of cultured neurons can induce apop-
ptotic actions of cytochrome c (Newmeyer and Green
tosis only when nerve growth factor is absent or added after the
injection of cytochrome c. This has led to the conclusion that 1998). Furthermore, it was concluded that such protec-
nerve growth factor and other neurotrophic factors lead to upregu-
tive molecules are expressed at low basal levels and up-
regulated within a short time after NGF exposure.
lation of an activity which directly interferes with the activation of
apoptosome activity and downstream caspases. In the absence of
Members of the IAP/ITA family are candidates for
neurotrophic factors, cytochrome c is released from mitochondria
by a process in which Bax is involved. This leads to the formation such protective proteins. They inhibit the activation of
of apoptosomes by a process also requiring Apaf-1 and ATP. By
procaspase-9 (Deveraux et al. 1998), which is initiated by
cytochrome c and Apaf-1 (Slee et al. 1999; Stennicke et
al. 1999). Furthermore, they inhibit the function of acti-
this means, procaspase-9 is cleaved and activated. Caspase-9 in
turn can cleave procaspase-3, -6 and/or -7, which then activate
downstream substrates, among them ICAD (inhibitor of caspase-
activated DNase, CAD) and finally leads to apoptosis of the neu- vated caspase-3, -6 and –7, and thus at least at two levels
ron. Members of the IAP family (inhibitor of apoptosis protein) interfere with cellular programs for apoptosis (Devereaux
can block activated caspases and inhibit these regulatory steps in
et al. 1997; Roy et al. 1997). The identification and clon-
the execution of apoptosis. Recent studies with cultured sensory
ing of the chick ita gene (Digby et al. 1996), which en-
and sympathetic neurons have shown that NGF and CNTF can
codes a protein of 611 amino acids with highest homolo-
lead to a more than 20-fold upregulation of ITA, the chicken ho-
mologue of cIAP-2 in cultured sensory and sympathetic neurons. gy to the human cIAP-2 gene (also called HIAP-1 or
This upregulation takes place within less than 6 h after addition of
MIHC), allowed us to investigate the involvement of this
the neurotrophic factor. Thus, ITA and mammalian homologues of
protein in NGF-mediated survival of developing chick
this protein could be essential mediators in the downstream signal-
sympathetic and sensory neurons. We were able to show
that NGF rapidly induced ITA expression in cultured
sympathetic and sensory neurons (Fig. 2). This upregula-
ing machinery for neurotrophic-factor-mediated survival of prima-
ry neurons
provide a second indication that members of the bcl-2 tion of ITA mRNA and protein levels involves the PI-3 K
family are involved, and that mitochondria play a role in pathway. Overexpression of ITA in primary sensory and
motoneuron cell death. Recently, it has been shown that sympathetic neurons can promote neuronal survival in the
BAX-dependent release of cytochrome c from mitochon- absence of NGF, and antisense expression of ITA can
dria plays an essential role in the initiation of cell death abolish the NGF survival effect in sensory and sympa-
in NGF-deprived sympathetic neurons (Deshmukh and thetic neurons. These actions are apparently mediated
Johnson Jr 1998). However, microinjection of cyto- through the baculovirus IAP repeat (BIR domains) of the
chrome c into the cytoplasm could not initiate cell death ITA protein, as expression of a BIR-deleted form of ITA
when the primary sympathetic neurons were grown in was without any effect on neuronal survival.

8
0
These data suggest that members of the IAP family, bodies in these mutant mice (Sagot et al. 1995). Similar
which include the mammalian IAP-2 and XIAP, are im- observations have also been made when pmn mice were
portant molecules involved in the signaling machinery treated with GDNF (Sagot et al. 1996). However, in con-
for neurotrophic-factor-mediated survival of sympathetic trast to CNTF, GDNF treatment does not lead to pro-
and sensory neurons. The expression of inhibitors for longed survival of the pmn mice. Although this neurotro-
caspases could be an essential mechanism which contrib- phic factor could reduce the extent of degeneration of
utes to motoneuron survival once these caspases are acti- motoneuron cell bodies, the degeneration of axons was
vated (Fig. 3), and they could be of considerable impor- not prevented, thus indicating that neurotrophic factors
tance in protecting motoneurons against any kind of pro- such as CNTF and GDNF differ in their potential to in-
apoptotic signaling which might occur during postnatal fluence specific parameters of motoneurons such as axon
life and which has to be neutralized for further mainte- maintenance or axon regeneration, at least in pmn mice.
nance of these cells.
During recent years accumulating evidence has also
suggested that nitric oxide (NO) can play a role in moto-
neuron cell death (Estevez et al. 1998a, 1998b). Depriva-
tion of embryonic motoneurons in culture from neurotro-
phic factors leads to enhanced production of neuronal ni-
tric acid synthetase. Moreover, enhanced nitrotyrosine
Pathological cell death of motoneurons
in postnatal animals
Despite significant progress in the understanding of sur- immunoreactivity could be observed in these motoneu-
vival and cell death signaling for developing motoneu- rons. NOS inhibitors could prevent apoptosis of cultured
rons, little is known about how these mechanisms relate motoneurons, at least during short culture periods of up
to postnatal cell death in human motoneuron disease. A to 3 days. Similar findings were made when a cell-
subtype of autosomal dominant familial amyotrophic lat- permeable scavenger of superoxide was added to neuro-
eral sclerosis is caused by mutations of the superoxide trophic-factor-deprived motoneurons in culture. It was
dismutase (SOD) gene (Rosen et al. 1993). The primary therefore suggested that enhanced superoxide and NO
function of SOD is to generate hydrogen peroxide from production, possibly also the production of peroxynitrite,
superoxide, and in a subsequent step hydrogen peroxide could play a role as proapoptotic signals, which repres-
is detoxified by glutathione peroxidase or catalase. ent a first step in the execution of cell death in develop-
These mutations have therefore been suggested to cause ing and postnatal motoneurons. This hypothesis is sup-
oxidative stress in motoneurons, thus leading to a slow ported by the observations that nitrotyrosine immunore-
degeneration process. However, SOD-1 enzyme activity activity is enhanced within a few hours in neurotrophic-
is not completely abolished, and several of the mutant factor-deprived motoneurons. Recently, it was demon-
forms of SOD-1 found in ALS patients show significant strated that NO mediates the cell death of motoneurons
or close to normal SOD-1 enzyme activity (Deng et al. when the superoxide-dismutase protein lacks a zinc ion,
1
993). Overexpression of mutated SOD-1 in motoneu- a scenario which could underlie familial ALS (Estevez et
rons causes cytoplasmic aggregation of the enzyme, and al. 1999). Nevertheless, very little is known so far about
neurons with such aggregates subsequently undergo apo- how these pathways are connected with activation of
ptotic cell death (Durham et al. 1997). Mice in which caspases in motoneurons, or whether these mechanisms
mutated SOD-1 (G93A) is overexpressed show a classi- represent an independent proapoptotic stimulus which
cal postnatal degeneration of motoneurons (Gurney et al. acts in a caspase-independent fashion.
1
994). Interestingly, when these mice are crossed with
bcl-2-overexpressing mice, onset of disease is delayed apoptotic stimuli was shown by crossbreeding mice car-
Kostic et al. 1997), suggesting that bcl-2 protects the rying the G93A mutation in the SOD gene with mice
First evidence for the interaction between these pro-
(
motoneurons from pathogenic processes caused by mu- overexpressing a dominant-negative ICE isoform which
tated SOD-1. Therefore, whatever the pathogenic pro- prevents caspases from activation of cell death pathways
cess in patients with mutations of SOD-1 or in transgenic (Friedlander et al. 1997). It thus will be interesting to
mice overexpressing mutated SOD-1 is, it seems to inter- know how neurotrophic factors interfere with such pro-
fere with classical cell death programs.
apoptotic pathways. This could be at the transcriptional
Similar findings have also been made with other mu- level, thus preventing the upregulation of proapoptotic
tant mice in which the underlying gene mutation is not genes such as NOS, or by upregulation of members of
known so far. Treatment with neurotrophic factors can the IAP family which block caspase activity. Future re-
interfere with motoneuron degeneration in mouse mu- search has to show how pro- and antiapoptotic signaling
tants such as progressive motoneuronopathy (pmn) in motoneurons is regulated in individual motoneurons,
(
1
Sendtner et al. 1992b) and wobbler (Mitsumoto et al. and how individual cell-specific genetic programs at spe-
994; Ikeda et al. 1995) mice, and it is not clear whether cific developmental stages and signals from contacting
the underlying gene defects are involved in cell death or cells such as muscle, glial cells and neurons function to-
survival signaling pathways. Interestingly, overexpres- gether in these regulatory processes.
sion of bcl-2 in pmn mice does not delay the onset of the
disease or prolong survival of pmn mice, although it in-
hibits the degeneration and cell death of motoneuron cell

8
1
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Deckwerth TL, Easton RM, Knudson CM, Korsmeyer SJ, Johnson
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