Elucidating the molecular mechanisms that underlie the target control of motoneuron death

Approximately half of the motoneurons generated during normal embryonic development undergo programmed cell death. Most of this death occurs during the time when synaptic connections are being formed between motoneurons and their target, skeletal muscle. Subsequent muscle activity stemming from this connection helps determine the final number of surviving motoneurons. These observations have given rise to the idea that motoneuron survival is dependent upon access to muscle derived trophic factors, presumably through intact neuromuscular synapses. However, it is not yet understood how the muscle regulates the supply of such trophic factors, or if there are additional mechanisms operating to control the fate of the innervating motoneuron. Recent observations have highlighted target independent mechanisms that also operate to support the survival of motoneurons, such as early trophic-independent periods of motoneuron death, trophic factors derived from Schwann cells and selection of motoneurons during pathfinding. Here we review recent investigations into motoneuron cell death when the molecular signalling between motoneurons and muscle has been genetically disrupted. From these studies, we suggest that in addition to trophic factors from muscle and/or Schwann cells, specific adhesive interactions between motoneurons and muscle are needed to regulate motoneuron survival. Such interactions, along with intact synaptic basal lamina, may help to regulate the supply and presentation of trophic factors to motoneurons.     

Developmental motoneuron cell death and neurotrophic factors

During the development of higher vertebrates, motoneurons are generated in excess. In the lumbar spinal cord of the developing rat, about 6000 motoneurons are present at embryonic day 14. These neurons grow out axons which make contact with their target tissue, the skeletal muscle, and about 50% of the motoneurons are lost during a critical period from embryonic day 14 until postnatal day 3. This process, which is called physiological motoneuron cell death, has been the focus of research aiming to identify neurotrophic factors which regulate motoneuron survival during this developmental period. Motoneuron cell death can also be observed in vitro when the motoneurons are isolated from the embryonic avian or rodent spinal cord. These isolated motoneurons and other types of primary neurons have been a useful tool for studying basic mechanisms underlying neuronal degeneration during development and under pathophysiological conditions in neurodegenerative disorders. Accumulating evidence from such studies suggests that some specific requirements of motoneurons for survival and proper function may change during development. The focus of this review is a synopsis of recent data on such specific mechanisms.     

Necdin protects embryonic motoneurons from programmed cell death

NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFα this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons.     

Developmental expression of voltage-dependent calcium currents in identified mouse motoneurons.

Previous work has shown that during chick embryonic development, large changes occur in the density of specific, motoneuronal calcium currents just prior to the period of naturally occurring motoneuron cell death. Here we report on calcium currents in mouse motoneurons isolated from embryos at the time of peak cell death and also during a subsequent developmental stage when supernumerary synapses are being eliminated. In mouse motoneurons, the density of high-voltage-activated calcium current increases significantly after the phase of cell death, during the period of synapse elimination.     

Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult.

Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-beta and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L-Cre; Stat3(flox/KO) mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.     

Cell death of motoneurons in the chick embryo spinal cord. VIII. Motoneurons prevented from dying in the embryo persist after hatching

A chronic neuromuscular blockade during those embryonic stages when naturally occurring spinal motoneuron death occurs, results in the prevention of this cell loss. The excess motoneurons are maintained as long as the neuromuscular blockade is continued; once embryonic neuromuscular activity resumes, however, the excess motoneurons undergo a delayed period of cell death. By contrast, the resumption of neuromuscular activity in these same preparations after hatching did not result in a delayed cell death. The excess motoneurons, prevented from dying in the embryo, persisted for as long as 4 days postnatally despite the presence of considerable limb motility. The maintenance of motoneurons may be regulated differently before and after hatching.     

Naturally occurring motoneuron cell death in rat upper respiratory tract motor nuclei: A histological,fast dil and immunocytochemical study in the nucleus ambiguus

The mammalian upper respiratory tract (URT) serves as the common modality for aspects of respiration, deglutition, and vocalization. Although these actions are dependent on coordinated and specific neuromuscular control, little is known about the development of URT control centers. As such, this study investigated the occurrence of naturally occurring motoneuron cell death (MCD) in the nucleus ambiguus (NA) of a developmental series of rats. Standard histological techniques were used to count motoneurons in the ventrolateral brainstem where the mature NA is found. In addition, the neural tracer, fast Dil, was used to determine whether motoneurons were still migrating into the region of the NA during the period that cell counts were first taken. Furthermore, to elucidate the potential effect of inadvertently counting large interneurons on the assessment of motoneuron numbers, an antibody to γ-aminobutyric acid (GABA) was used. The results of this study have, for the first time, demonstrated that MCD occurs in a URT-related motor nucleus. Approximately a 50% cell death was observed during the prenatal development of NA, with no further loss seen postnatally. The fast DiI studies showed that by embryonic day 17, NA was fully formed, suggesting that motoneuron migration from the basal plate was complete. In addition, use of the GABA antibody showed a lack of inhibitory interneurons within the NA. The finding of MCD in the NA helps define a critical period in the formation of URT neuromuscular control. As the course of MCD is modifiable by epigenetic signals, insult to the organism during this prenatal period may compromise future URT control. © 1995 John Wiley & Sons, Inc.     

The response of motoneurons to neurotrophins

The ongoing search for neurotrophic factors for motoneurons has led to the identification of a number of molecules which regulate motoneuron survival and function. Among these factors, the neurotrophins brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5 but not nerve growth factor (NGF), can prevent embryonic and postnatal motoneuron cell death in a variety of experimental paradigms. Analysis of expression of p75, trkB and trkC—components of the neurotrophin receptors—supports a potential physiological role for these factors as muscle- and glial-derived trophic factors for motoneurons. However, the survival of motoneurons during embryonic development is not reduced in the absence of BDNF, NT-3 or NT-4, as revealed by gene knockout experiments. This points to the involvement of additional trophic factors in the regulation of embryonic and postnatal motoneuron survival. The purpose of this review is to bring together the often prophetic observations from earlier studies—prior to the identification and characterization of these neurotrophins—with more recent results. Special issue dedicated to Dr. Hans Thoenen.     

Chronic application of curare does not increase the level of motoneuron survival-promoting activity in limb muscle extracts during the naturally occurring motoneuron cell death period

Naturally occurring motoneuron cell death during development is a well-described phenomenon and the existence of a survival factor provided by target muscles has been postulated. Blockade of activity by chronic application of a neuromuscular junction blocker rescues almost all motoneurons from cell death. The present study was conducted in order to examine the possibility that the motoneuron survival-promoting activity in muscles increases following activity blockade. Cell culture was used to assess the degree of motoneuron survival-promoting activity present in muscle extracts. Embryonic chick motoneurons were labeled by injecting the water-insoluble fluorescent dye, DiI (Molecular Probes, Inc.) into the spinal nerves both before and during the cell death period. The labeled cells extending long neurites were counted after 2 days of culture as viable motoneurons in low-density heterogeneous cell cultures. The culture medium, Ham F12/DMEM (1:1 mixture) supplemented with 10% horse serum, 5% chick serum, and 5% fetal calf serum, was employed as a basic culture medium for assessing motoneuron survival factor, since it supported the survival of a significantly higher number of motoneurons derived from embryos before cell death than those during the cell death period, thus representing the motoneuron's requirement for survival factor in vivo. The number of surviving motoneurons clearly increased in proportion to the amount of muscle extract added to the culture medium. In comparison with control chick embryos, the dose-response relation between the number of surviving motoneurons and the amount of muscle extract added did not change when embryos were used after chronic application of curare. These results therefore indicate that survival factor derived from target muscle is crucial to the in vitro motoneurons during the cell death period, but do not support the idea that inactive muscle contains a higher amount of the survival factor.     

Regulation of motoneuron death in the spinal nucleus of the bulbocavernsus

A sexual dimorphism in the number of motoneurons in the spinal nucleus of the bulbocavernosus (SNB) of rats is engendered by a sex difference in ontogenetic cell death. Testicular secretions, specifically androgenic steroids, reduce SNB motoneuron death in males. The fate of the target muscles generally mirrors that of the motoneurons, and androgens appear to exert their effects upon the target muscles, sparing the motoneurons as a secondary consequence. Treatment with ciliary neurotrophic factor can also spare SNB motoneurons in newborn females, raising the possibility that this factor normally mediates androgen's effect upon motoneuron survival. The ontogeny of calcitonin gene-related peptide immunoreactivity is delayed in SNB cells compared with other motoneurons and is further delayed in the SNB cells of females. In both sexes, calcitonin gene-related peptide is detected after the period of SNB motoneuron death is complete. A sex difference in motoneuron number is also seen in the human homologue of the SNB and, because ontogenetic death of motoneurons in humans overlaps the period of androgen secretion, may arise in a manner similar to that in the rat SNB. © 1992 John Wiley & Sons, Inc.     

Interactions between spinal cord stimulation and activity blockade in the regulation of synaptogenesis and motoneuron survival in the chick embryo

The present study investigated the effects of spinal cord stimulation, neuromuscular blockade, or a combination of the two on neuromuscular development both during and after the period of naturally occurring motoneuron death in the chick embryo. Electrical stimulation of the spinal cord was without effect on motoneuron survival, synaptogenesis, or muscle properties. By contrast, activity blockade rescued motoneurons from cell death and altered synaptogenesis. A combination of spinal cord stimulation and activity blockade resulted in a marked increase in motoneuron death, and also altered synaptogenesis similar to that seen with activity blockade alone. Perturbation of normal nerve–muscle interactions by activity blockade may increase the vulnerability of developing motoneurons to excessive excitatory afferent input (spinal cord stimulation) resulting in excitotoxic-induced cell death. © 1993 John Wiley & Sons, Inc.     

Naturally occurring motoneuron cell death in rat upper respiratory tract motor nuclei: A histological,fast Dil and immunocytochemical study in the hypoglossal nucleus

We have previously reported on our investigation of motoneuron cell death (MCD) in the rat nucleus ambiguus (NA). This article focuses on the other major upper respiratory tract motor nucleus: the hypoglossal. The hypoglossal nucleus (XII) contains motoneurons to the tongue and, as such, plays a critical role in defining patterns of respiration, deglutition, and vocalization. Motoneuron counts were made in XII in a developmental series of rats. In addition, the neural tracer fast DiI was used to ensure that all hypoglossal motoneurons had migrated into the nucleus at the time cell death was assessed. Furthermore, an antibody to γ-aminobutyric acid (GABA) was used to determine the potential effect of inadvertently counting large interneurons on motoneuron counts. Cell death in XII was shown to occur entirely prenatally with a loss of 35% of cells between embryonic day 16 (E16) and birth. Fast DiI tracings of the prenatal hypoglossal nerve indicated that all motoneurons were present in a well-defined nucleus by E15. Immunocytochemical staining for GABA demonstrated considerably fewer interneurons than motoneurons in XII. These findings in XII, in comparison with those previously reported for NA, demonstrate differences in the timing and amount of cell death between upper respiratory tract motor nuclei. These differences establish periods during which one nucleus may be preferentially insulted by environmental or teratogenic factors. Preferential insults may underlie some of the upper respiratory tract incoordination pathologies seen in the newborn such as the sudden infant death syndrome (SIDS). © 1995 John Wiley & Sons, Inc.     

Effects of a protein synthesis inhibitor on the hormonally mediated regression and death of motoneurons in the tobacco hornworm,Manduca sexta

The larval–pupal transformation of Manduca sexta is accompanied by the loss of the abdominal prolegs. The proleg muscles degenerate, the dendritic arbors of proleg motoneurons regress, and a subset of the proleg motoneurons dies. The regression and death of proleg motoneurons are triggered by the prepupal peak of ecdysteroids in the hemolymph. To investigate the possible involvement of protein synthesis in these events, we gave insects repeated injections of the protein synthesis inhibitor, cycloheximide (CHX), during the prepupal peak. Examination of insects 3–5 days following CHX treatment showed that CHX inhibited the death of proleg motoneurons and the production of pupal cuticle in a dose-dependent fashion. When insects were allowed to survive for 10 days after the final CHX injection, motoneuron death and pupal cuticle production sometimes occurred belatedly, apparently in response to the ecdysteroid rise that normally triggers adult development. CHX treatments that inhibited motoneuron death were less effective in inhibiting dendritic regression in the same neurons. In another set of experiments, abdomens were isolated from the ecdysteroid-secreting glands prior to the prepupal peak, and infused with 20-hydroxyecdysone (20-HE). Single injections of CHX delivered just prior to the start of the 20-HE infusion inhibited motoneuron death and pupal cuticle production, but in the range of doses tested, did not prevent dendritic regression. Our findings suggest that protein synthesis is a required step in the steroid-mediated death of proleg motoneurons, and that dendritic regression is less susceptible to inhibition by CHX than is motoneuron death. © 1993 John Wiley & Sons, Inc.     

Programmed cell death of embryonic motoneurons triggered through the Fas death receptor

About 50% of spinal motoneurons undergo programmed cell death (PCD) after target contact, but little is known about how this process is initiated. Embryonic motoneurons coexpress the death receptor Fas and its ligand FasL at the stage at which PCD is about to begin. In the absence of trophic factors, many motoneurons die in culture within 2 d. Most (75%) of these were saved by Fas-Fc receptor body, which blocks interactions between Fas and FasL, or by the caspase-8 inhibitor tetrapeptide IETD. Therefore, activation of Fas by endogenous FasL underlies cell death induced by trophic deprivation. In the presence of neurotrophic factors, exogenous Fas activators such as soluble FasL or anti-Fas antibodies triggered PCD of 40-50% of purified motoneurons over the following 3-5 d; this treatment led to activation of caspase-3, and was blocked by IETD. Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant. Levels of Fas expressed by motoneurons varied little, but FasL was upregulated in the absence of neurotrophic factors. Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation. Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations.     

Characterization of the execution pathway of developing motoneurons deprived of trophic support

Avian spinal motoneurons have been well characterized with regard to developmental programmed cell death (PCD). Approximately 50% of the neurons originally generated undergo cell death as they innervate their target muscles, and target derived trophic support plays an important role in regulating survival of these neurons. To investigate events mediating motoneuron PCD, we have examined the role of Bcl-2 family proteins, cytochrome C, and caspase-9 in this process. We report that while protein levels of Bcl-2, Bcl-xL, and Bax do not change within motoneurons as they become committed to die, a translocation of Bax from the cytosol to organelle membranes and the nucleus occurs coincident with the time when motoneurons become committed to cell death. In addition, cytochrome C is released from mitochondria to the cytosol in dying cells prior to the activation of caspases. Consequently, an enhanced caspase-9-like activity was detected in dying cells, and this activity was upstream and necessary for the appearance of a caspase-3-like activity. These results allow us to further define some of the critical events that mediate the execution phase of motoneuron death following trophic factor deprivation.     

Brain-derived proteins that rescue spinal motoneurons from cell death in the chick embryo: Comparisons with target-derived and recombinant factors

Spinal motoneurons that normally die during early development can be rescued by a variety of purified growth or neurotrophic factors and target tissue extracts. There is also indirect evidence that brain or supraspinal afferent input may influence lumbar motoneuron survival during development and that this effect may be mediated by central nervous system–derived trophic agents. This report examines the biological and biochemical properties of motoneuron survival activity obtained from extracts of the embryonic chick brain. Treatment with an ammonium sulfate (25% to 75%) fraction of embryonic day 16 (E16) or E10 brain extracts rescued many spinal motoneurons that otherwise die during the normal period of cell death in vivo (E6 to E10). The same fractions also enhanced lumbar motoneuron survival following deafferentation. There were both similarities and differences between the active fractions derived from brain extracts (BEX) when compared with extracts derived from target muscles (MEX) or with purified neurotrophic factors. Survival activity from E10 BEX was as effective in promoting motoneuron survival as E10 MEX and more effective than astrocyte-conditioned media. Unlike MEX, the active fractions from BEX also rescued placode-derived nodose ganglion cells. In addition, unlike nerve growth factor and brain-derived neurotrophic factor, active BEX fractions did not rescue neural crest-derived dorsal root ganglion cells or sympathetic ganglion neurons. Interestingly, among many cranial motor and other brainstem nuclei examined, only the survival of motoneurons from the abducens nucleus was enhanced by BEX. Active proteins obtained from BEX were further separated by gel filtration chromatography and by preparative isolelectric focusing techniques. Activity was recovered in a basic (pI8) and an acidic (pI5) small molecular weight protein fraction (20 kD or less). The specific activity of the basic fraction was increased ×66 when compared with the specific activity of crude BEX, and the basic fraction had a slightly higher specific activity than the acidic fraction. The biological and biochemical properties of these fractions are discussed in the context of known neurotrophic factors and their effects on normal and lesion-induced motoneuron death during development. © 1995 John Wiley & Sons, Inc.