Reduction of naturally occurring motoneuron death in vivo by a target-derived neurotrophic factor.

Treatment of chick embryos in ovo with crude and partially purified extracts from embryonic hindlimbs (days 8 to 9) during the normal cell death period (days 5 to 10) rescues a significant number of motoneurons from degeneration. The survival activity of partially purified extract was dose-dependent and developmentally regulated. The survival of sensory, sympathetic, parasympathetic, and a population of cholinergic sympathetic preganglionic neurons was unaffected by treatment with hindlimb extract. The massive motoneuron death that occurs after early target (hindlimb) removal was partially ameliorated by daily treatment with the hindlimb extract. These results indicate that a target-derived neurotrophic factor is involved in the regulation of motoneuron survival in vivo.

Control of embryonic motoneuron survival in vivo by ciliary neurotrophic factor.

During development of the nervous system, neurons in many regions are overproduced by proliferation, after which the excess cells are eliminated by cell death. The survival of only a proportion of neurons during normal development is thought to be regulated by the limited availability of neurotrophic agents. One such putative trophic agent is ciliary neurotrophic factor (CNTF), a polypeptide that promotes the survival of ciliary, sensory, and sympathetic neurons in vitro. In contrast to the results of in vitro studies, however, the daily treatment of chick embryos in vivo with purified human recombinant CNTF failed to rescue any of these cell populations from cell death, whereas CNTF did promote the in vivo survival of spinal motoneurons. Thus, CNTF may not act as a neurotrophic agent in vivo for those embryonic neurons (especially ciliary neurons) on which it acts in vitro. Rather, CNTF may be required for in vivo survival of motoneurons.

Rescue of motoneurons from cell death by a purified skeletal muscle polypeptide: effects of the ChAT development factor, CDF.

Rat skeletal muscle contains a 22 kd polypeptide that increases the level of choline acetyltransferase (ChAT) activity in cultures of embryonic rat spinal cord neurons and has been purified to homogeneity. The application of this factor, ChAT development factor or CDF, to developing chick embryos during the period of naturally occurring motoneuron cell death significantly increased the survival of motoneurons but did not affect the survival of dorsal root ganglion neurons or sympathetic preganglionic neurons (column of Terni). These results provide the first demonstration that an isolated, skeletal muscle-derived molecule can selectively enhance the survival of motoneurons in vivo and suggest that CDF may function in vivo to regulate the survival and development of motoneurons.

Positive and negative effects of neurotrophins on the isthmo-optic nucleus in chick embryos.

The survival of neurons in the developing isthmo-optic nucleus (ION) is believed to depend on the retrograde transport of trophic molecules from the target, the contralateral retina. We now show that ION neurons transport nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) retrogradely and that BDNF and NT-3 support the survival of ION neurons in vivo and promote neurite outgrowth in vitro. Surprisingly, NGF enhanced normal developmental cell death in vivo in a dose-dependent way. These findings show that increased levels of NGF can have adverse effects on differentiated neurons. The negative effect of NGF could be mimicked by intraocular injection of antibodies that block binding of neurotrophins to the 75 kd neurotrophin receptor (p75). These data implicate a role for the p75 receptor in NGF's neurotoxicity and indicate that this receptor is involved in the mechanism by which ION neurons respond to BDNF and NT-3 in the target.

Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro.

Members of the CED-3/interleukin-1 beta-converting enzyme (ICE) protease family have been implicated in cell death in both invertebrates and vertebrates. In this report, we show that peptide inhibitors of ICE arrest the programmed cell death of motoneurons in vitro as a result of trophic factor deprivation and in vivo during the period of naturally occurring cell death. In addition, interdigital cells that die during development are also rescued in animals treated with ICE inhibitors. Taken together, these results provide the first evidence that ICE or an ICE-like protease plays a regulatory role not only in vertebrate motoneuron death but also in the developmentally regulated deaths of other cells in vivo.

Hepatocyte growth factor/scatter factor is a neurotrophic survival factor for lumbar but not for other somatic motoneurons in the chick embryo.

Hepatocyte growth factor/scatter factor (HGF/SF) is expressed in the developing limb muscles of the chick embryo during the period of spinal motoneuron (MN) programmed cell death, and its receptor c-met is expressed in lumbar MNs during this same period. Although cultured motoneurons from brachial, thoracic, and lumbar segments are all rescued from cell death by chick embryo muscle extract (CMX) as well as by other specific trophic agents, HGF/SF only promotes the survival of lumbar MNs. Similarly, treatment of embryos in ovo with exogenous HGF/SF rescues lumbar but not other somatic MNs from cell death. Blocking antibodies to HGF/SF (anti-HGF) reduce the effects of CMX on MN survival in vitro and decrease the number of lumbar MNs in vivo. The expression of c-met on MNs in vivo is regulated by a limb-derived trophic signal distinct from HGF/SF. HGF/SF is a potent, select, and physiologically relevant survival factor for a subpopulation of developing spinal MNs in the lumbar segments of the chick embryo.

Glial cell line-derived neurotrophic factor and developing mammalian motoneurons: regulation of programmed cell death among motoneuron subtypes.

Because of discrepancies in previous reports regarding the role of glial cell line-derived neurotrophic factor (GDNF) in motoneuron (MN) development and survival, we have reexamined MNs in GDNF-deficient mice and in mice exposed to increased GDNF after in utero treatment or in transgenic animals overexpressing GDNF under the control of the muscle-specific promoter myogenin (myo-GDNF). With the exception of oculomotor and abducens MNs, the survival of all other populations of spinal and cranial MNs were reduced in GDNF-deficient embryos and increased in myo-GDNF and in utero treated animals. By contrast, the survival of spinal sensory neurons in the dorsal root ganglion and spinal interneurons were not affected by any of the perturbations of GDNF availability. In wild-type control embryos, all brachial and lumbar MNs appear to express the GDNF receptors c-ret and GFRalpha1 and the MN markers ChAT, islet-1, and islet-2, whereas only a small subset express GFRalpha2. GDNF-dependent MNs that are lost in GDNF-deficient animals express ret/GFRalpha1/islet-1, whereas many surviving GDNF-independent MNs express ret/GFRalpha1/GFRalpha2 and islet-1/islet-2. This indicates that many GDNF-independent MNs are characterized by the presence of GFRalpha2/islet-2. It seems likely that the GDNF-independent population represent MNs that require other GDNF family members (neurturin, persephin, artemin) for their survival. GDNF-dependent and -independent MNs may reflect subtypes with distinct synaptic targets and afferent inputs.

Programmed cell death of developing mammalian neurons after genetic deletion of caspases.

An analysis of programmed cell death of several populations of developing postmitotic neurons after genetic deletion of two key members of the caspase family of pro-apoptotic proteases, caspase-3 and caspase-9, indicates that normal neuronal loss occurs. Although the amount of cell death is not altered, the death process may be delayed, and the cells appear to use a nonapoptotic pathway of degeneration. The neuronal populations examined include spinal interneurons and motor, sensory, and autonomic neurons. When examined at both the light and electron microscopic levels, the caspase-deficient neurons exhibit a nonapoptotic morphology in which nuclear changes such as chromatin condensation are absent or reduced; in addition, this morphology is characterized by extensive cytoplasmic vacuolization that is rarely observed in degenerating control neurons. There is also reduced terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling in dying caspase-deficient neurons. Despite the altered morphology and apparent temporal delay in cell death, the number of neurons that are ultimately lost is indistinguishable from that seen in control animals. In contrast to the striking perturbations in the morphology of the forebrain of caspase-deficient embryos, the spinal cord and brainstem appear normal. These results are consistent with the growing idea that the involvement of specific caspases and the occurrence of caspase-independent programmed cell death may be dependent on brain region, cell type, age, and species or may be the result of specific perturbations or pathology.

Cardiotrophin-1, a muscle-derived cytokine, is required for the survival of subpopulations of developing motoneurons.

Developing motoneurons require trophic support from their target, the skeletal muscle. Despite a large number of neurotrophic molecules with survival-promoting activity for isolated embryonic motoneurons, those factors that are required for motoneuron survival during development are still not known. Cytokines of the ciliary neurotrophic factor (CNTF)-leukemia inhibitory factor (LIF) family have been shown to play a role in motoneuron (MN) survival. Importantly, in mice lacking the LIFRbeta or the CNTFRalpha there is a significant loss of MNs during embryonic development. Because genetic deletion of either (or both) CNTF or LIF fails, by contrast, to perturb MN survival before birth, it was concluded that another ligand exists that is functionally inactivated in the receptor deleted mice, resulting in MN loss during development. One possible candidate for this ligand is the CNTF-LIF family member cardiotrophin-1 (CT-1). CT-1 is highly expressed in embryonic skeletal muscle, secreted by myotubes, and promotes the survival of cultured embryonic mouse and rat MNs. Here we show that ct-1 deficiency causes increased motoneuron cell death in spinal cord and brainstem nuclei of mice during a period between embryonic day 14 and the first postnatal week. Interestingly, no further loss was detectable during the subsequent postnatal period, and nerve lesion in young adult ct-1-deficient mice did not result in significant additional loss of motoneurons, as had been previously observed in mice lacking both CNTF and LIF. CT-1 is the first bona fide muscle-derived neurotrophic factor to be identified that is required for the survival of subgroups of developing motoneurons.

Characterization of spinal motoneuron degeneration following different types of peripheral nerve injury in neonatal and adult mice.

Experimental lesions have been used widely to induce motoneuron (MN) degeneration as a model to test the ability of different trophic molecules to prevent lesion-induced alterations. However, the morphological mechanisms of spinal MN death following different types of lesions is not clear at the present time. In this study, we have characterized the morphological characteristics of MN cell death by examining DNA fragmentation and the ultrastructural and light microscopic morphological features of MNs following different types of spinal nerve injury (i.e., axotomy and avulsion) in the developing and adult mouse. In neonatal mice, axotomy induced cell death as well as the atrophy of MNs that survived the injury. DNA fragmentation could be detected by using the terminal deoxynucleotidyl transferase (TUNEL) method during the cell death process following neonatal axotomy, whereas TUNEL labeling was not observed following either neonatal or adult avulsion. However, with the exception of TUNEL labeling, the morphological characteristics of MN death following neonatal axotomy and avulsion were similar, and both resembled most closely the form of programmed cell death termed cytoplasmic or type 3B, which exhibits similarities as well as differences with currently accepted definitions of apoptosis. By contrast, adult avulsion resulted in a type of degeneration that resembled necrosis more closely. However, even there, the morphology was mixed, showing characteristics of both apoptosis and necrosis. These results indicate that the mode of MN degeneration is complex and is related to developmental age and type of lesion.

Neuromuscular development in the avian paralytic mutant crooked neck dwarf (cn/cn): further evidence for the role of neuromuscular activity in motoneuron survival.

Neuromuscular transmission and muscle activity during early stages of embryonic development are known to influence the differentiation and survival of motoneurons and to affect interactions with their muscle targets. We have examined neuromuscular development in an avian genetic mutant, crooked neck dwarf (cn/cn), in which a major phenotype is the chronic absence of the spontaneous, neurally mediated movements (motility) that are characteristic of avian and other vertebrate embryos and fetuses. The primary genetic defect in cn/cn embryos responsible for the absence of motility appears to be the lack of excitation-contraction coupling. Although motility in mutant embryos is absent from the onset of activity on embryonic days (E) 3-4, muscle differentiation appears histologically normal up to about E8. After E8, however, previously separate muscles fuse or coalesce secondarily, and myotubes exhibit a progressive series of histological and ultrastructural degenerative changes, including disarrayed myofibrils, dilated sarcoplasmic vesicles, nuclear membrane blebbing, mitochondrial swelling, nuclear inclusions, and absence of junctional end feet. Mutant muscle cells do not develop beyond the myotube stage, and by E18-E20 most muscles have almost completely degenerated. Prior to their breakdown and degeneration, mutant muscles are innervated and synaptic contacts are established. In fact, quantitative analysis indicates that, prior to the onset of muscle degeneration, mutant muscles are hyperinnervated. There is increased branching of motoneuron axons and an increased number of synaptic contacts in the mutant muscle on E8. Naturally occurring cell death of limb-innervating motoneurons is also significantly reduced in cn/cn embryos. Mutant embryos have 30-40% more motoneurons in the brachial and lumbar spinal cord by the end of the normal period of cell death. Electrophysiological recordings (electromyographic and direct records form muscle nerves) failed to detect any differences in the activity of control vs. mutant embryos despite the absence of muscular contractile activity in the mutant embryos. The alpha-ryanodine receptor that is genetically abnormal in homozygote cn/cn embryos is not normally expressed in the spinal cord. Taken together, these data argue against the possibility that the mutant phenotype described here is caused by the perturbation of a central nervous system (CNS)-expressed alpha-ryanodine receptor. The hyperinnervation of skeletal muscle and the reduction of motoneuron death that are observed in cn/cn embryos also occur in genetically paralyzed mouse embryos and in pharmacologically paralyzed avian and rat embryos. Because a primary common feature in all three of these models is the absence of muscle activity, it seems likely that the peripheral excitation of muscle by motoneurons during normal development is a major factor in regulating retrograde muscle-derived (or muscle-associated) signals that control motoneuron differentiation and survival.

Effects of neurotrophic factors on motoneuron survival following axonal injury in newborn rats.

Using two different lesion models, the spinal root avulsion and the distal nerve axotomy, the present study investigated effects of known neurotrophic factors on motoneuron survival in newborn rats. Results of the present study show that 100% of motoneurons in the lesioned spinal segment die at 1 week following root avulsion, and more than 80% of them die at 2 weeks following distal nerve axotomy. Local application of GDNF can rescue 92% of motoneurons up to 1 week from degeneration due to root avulsion and almost 100% of them up to 2 weeks from degeneration due to distal nerve axotomy. Local application of BDNF fails to prevent any motoneuron death in newborn rats following root avulsion, but it can rescue about 50% of motoneurons up to 2 weeks from degeneration due to distal nerve axotomy. CNTF and IGF-1 fail to prevent any motoneuron death following either distal nerve axotomy or root avulsion. Thus, comparing all the neurotrophic factors tested in this study, GDNF is most effective in preventing death of motoneurons following axonal injury in newborn rats.

Enhancement of naturally occurring cell death in the sympathetic and parasympathetic ganglia of the chicken embryo following blockade of ganglionic transmission.

Both presynaptic and postsynaptic blockade of ganglionic transmission during the period of naturally occurring ganglion cell death reduced the number of surviving neurons in the sympathetic ganglia (SG) and ciliary ganglion (CG). The CG was chosen for analysis because there was a temporal separation between cell proliferation and death in the CG but not in the SG. Ganglion cell proliferation and migration were unaffected by ganglionic blockade. The increased ganglion cell loss that followed ganglionic blockade was accompanied by an increased number of degenerating cells. These results indicate that the decreased number of healthy ganglion cells that followed ganglionic blockade was the result of enhanced naturally occurring cell death.

Long-term effects of a single dose of brain-derived neurotrophic factor on motoneuron survival following spinal root avulsion in the adult rat.

The long-term effect of a single dose of Brain-derived neurotrophic factor (BDNF) treatment on adult motoneuron survival and on expression of nitric oxide synthase (NOS) following nerve injury (avulsion) was investigated and compared with that of continuous BDNF treatment. By 6 weeks post-injury, more than 80% of motoneurons survived in animals treated with either a single dose or continuous treatment of BDNF, while only 30% of motoneurons survived in control animals (avulsion only). There were no significant differences in motoneuron survival between animals receiving a single dose and those with continuous treatment of BDNF. Additionally, the expression of NOS in avulsed motoneurons was almost completely inhibited in all BDNF treatment groups regardless of the mode of administration (single vs. continuous). These data indicate that treatment with a single dose of BDNF at the time of injury can inhibit NOS expression and provide the first evidence that in this situation BDNF has a long-term rescue effect on adult motoneuron survival after root avulsion.

Naturally-occurring neuron death in the ciliary ganglion of the chick embryo following removal of preganglionic input: evidence for the role of afferents in ganglion cell survival.

With only a few exceptions, most investigations of the mechanisms involved in naturally-occurring neuron death have focused on interactions between neurons and their targets, with much less attention having been paid to the possible role of the afferent inputs in this phenomenon. This is true of the avian ciliary ganglion (CG), which is composed of a population of peripheral autonomic neurons that project to smooth and striated musculature in the eye and which receive afferents from a single source, the accessory oculomotor nucleus (AON), which is the avian homolog of the Edinger-Westphal nucleus. Although several lines of evidence strongly support the important role of targets in regulating the death and survival of CG neurons, the role of afferents has not yet been systematically examined. Following the destruction of the AON on embryonic day (E) 4, which is several days before the onset of normal cell death in the CG, we have found that by the end of the normal cell death period (E14-E15), 85-90% of the CG neurons degenerate and die, compared to 50% in controls. This is comparable to the amount of induced cell loss that occurs following removal of the optic vesicle containing the CG targets. The neurons surviving after deafferentation appear to be sustained by some influence from their targets since combined deafferentation and eye removal results in the loss of virtually all neurons in the CG. Following deafferentation of the CG on E4, the ganglion develops normally up to about E10, after which a precipitous loss of cells occurs. Based on several kinds of evidence (e.g., axon counts, silver stain, retrograde labeling of the CG), we conclude that the deafferented neurons project to and innervate their muscular targets in the eye. Therefore, the increased cell death following deafferentation cannot be due to the failure of deafferented neurons to contact their targets. The deafferented neurons undergo a normal sequence of initial ultrastructural differentiation. When they do begin to degenerate, the type of fine structural changes they exhibit appears indistinguishable from the degenerative changes observed in control embryos. Neurons in deafferented ganglia were occasionally observed to receive synaptic contacts, which we attribute to aberrant intraganglionic connections induced by deafferentation. These contacts probably play little, if any, role in the maintenance of neurons since, as noted above, following combined deafferentation and target deletion virtually all neurons degenerate and die.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell death of motoneurons in the chick embryo spinal cord. XI. Acetylcholine receptors and synaptogenesis in skeletal muscle following the reduction of motoneuron death by neuromuscular blockade.

Treatment of chick embryos with neuromuscular blocking agents such as curare during periods of naturally occurring motoneuron death results in a striking reduction of this normal cell loss. Inactivity-induced changes in motoneuron survival were found to be associated with increased levels of AChRs and AChR-clusters in skeletal muscle and with increased focal sites of AChE that are innervated ('synaptic sites'). Treatment of embryos with curare after the normal cell death period (E12-E15) resulted in no change in motoneuron survival. Although AChR-clusters and focal sites of AChE were increased in these embryos on E16, many of these sites were uninnervated. Treatment of embryos with nicotine or decamethonium (E6-E10) also reduced neuromuscular activity but did not alter motoneuron survival nor did such treatment alter AChRs. The different effects of curare vs nicotine and decamethoniam on motoneuron survival and AChRs may be related to the fact that the former is a competitive blocker whereas the latter two drugs are depolarizing blockers. Finally, treatment of embryos (E6-9) with doses of curare (1 mg daily) that allow for the almost complete recovery of neuromuscular activity a few days following treatment (by E16) resulted in the gradual loss of the excess motoneurons that were present on E10, and by E16 the number of remaining AChR clusters and focal sites of AChE were also decreased to levels comparable to control values. Inactivity-induced changes in AChRs or AChR-clusters may be an important factor in the reduced motoneuron death that accompanies neuromuscular blockade during critical stages of development. These receptor changes very likely reflect increased synaptogenesis in the muscles of paralyzed embryos which in turn may act to reduce motoneuron death by providing increased access to muscle-derived neurotrophic molecules.

The lack of effect of basic and acidic fibroblast growth factors on the naturally occurring death of neurons in the chick embryo.

In vivo treatment of developing chick embryos with acidic and basic fibroblast growth factors (aFGF and bFGF) failed to affect the differentiation and survival of several populations of developing neurons in the CNS and PNS. All of the neuronal populations examined are known to undergo naturally occurring cell death, and they include spinal and cranial motoneurons, dorsal root ganglia, sympathetic ganglia, nodose ganglia, ciliary ganglia, and sympathetic preganglionic neurons in the PNS, as well as the accessory oculomotor nucleus, the isthmo-optic nucleus, and the brainstem auditory nuclei laminaris and magnocellularis in the CNS. Despite the lack of effect of bFGF on neuronal survival and differentiation, in vivo treatment increased the serum levels of bFGF and stimulated the proliferation of non-neuronal cells in the spinal cord. Therefore, although the administration of exogenous FGF to the developing chick embryo in vivo clearly has some biological activity in the CNS, it was nonetheless ineffective in promoting neuronal survival or differentiation. These data do not support the idea that FGF is a physiologically relevant neurotrophic agent in the developing avian nervous system.

Early regional variations in motoneuron numbers arise by differential proliferation in the chick embryo spinal cord.

Regional differences in the number of motoneurons in the spinal cord of the chick are thought to arise developmentally by region-specific cell death and cell migration. In this way, a numerically homogeneous motor column throughout the spinal cord is believed to be molded into the adult pattern. Region-specific differences in proliferation are not thought to play a significant role in this process. By counting motoneurons in serial sections throughout the rostral-caudal extent of the spinal cord on Embryonic Day 4 in the chick, we have found that the numerical variations in motoneurons in different spinal cord regions are already foreshadowed by this stage, which is before the onset of both cell death and the secondary migration of neurons out of the motor column. These results indicate that although nonproliferative events may contribute to the later regional variations in motoneuron numbers, the initial differences themselves are created early on by regionally specific proliferative events.

Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: evidence for the role of cell death genes.

Treatment of chick embryos in ovo for 10-12 hr with inhibitors of protein and RNA synthesis during the peak time of normal cell death (Embryonic Day 8) for motoneurons and dorsal root ganglion cells markedly reduces the number of degenerating neurons in these populations. The massive neuronal death induced by the early absence of the limbs was also blocked almost completely by these agents. Further, the death of neurons following peripheral axotomy at the end of the normal cell death period (Embryonic Day 10) was reduced significantly by treatment with inhibitors of biosynthetic reactions. These results indicate that, in vivo, naturally occurring neuronal death, neuronal death induced by the absence of peripheral targets, and axotomy-induced neuronal death later in development all require active gene expression and protein and RNA synthesis. Therefore, neuronal death in a variety of situations may reflect the expression of a developmental fate that can normally only be overridden or suppressed by specific environmental signals (e.g., neurotrophic molecules).

Target contact regulates expression of synaptotagmin genes in spinal motor neurons in vivo.

During neuromuscular development, neuronal contact with peripheral targets is associated with an increase in synaptic vesicle protein (SVP) gene expression, suggesting that target contact and upregulation of SVP genes are causally related. To test this idea, we analyzed the developmental expression pattern of synaptotagmin (syt) mRNAs in the chick lateral motor column (LMC) using in situ hybridization. Syt I mRNA in the LMC is upregulated from Embryonic Day 4.5 (E4.5) to E5.5, coincident with the time these neurons begin to make contact with their muscle targets. In contrast, levels of mRNA for neurofilament do not change during this time. Extirpation of the limb bud prior to motor axon outgrowth eliminates the increase in syt I mRNA ipsilaterally. Later in development, there is a switch in syt isoform abundance in the LMC, with syt II mRNA being upregulated between E15 and E20 and syt I mRNA being downregulated. Our results suggest that contact with targets upregulates syt I gene expression during neuromuscular synapse formation in vivo, and that a later stage of synaptic maturation involves changes in SVP isoform abundance.