Characterization of early pathogenesis in the SOD1(G93A) mouse model of ALS: part I, background and methods.

Charcot first described amyotrophic lateral sclerosis (ALS) in 1869; however, its causes remain largely unknown and effective, long-term treatment strategies are not available. The first mouse model of ALS was developed after the identification of mutations in the superoxide dismutase 1 (SOD1) gene in 1993, and accordingly most of our knowledge of the etiology and pathogenesis of the disease comes from studies carried out using this animal model. Although numerous preclinical trials have been conducted in the mutant SOD1 mouse models, the results have been disappointing because they did not positively translate to clinical trials. One explanation may be that current understanding of when and where pathogenesis begins is insufficient to accurately guide preclinical trials. Further characterization of these early events may provide insight into disease onset, help in the discovery of presymptomatic diagnostic disease markers, and identify novel therapeutic targets. Here, we describe the rationale, approach, and methods for our extensive analysis of early changes that included an ultrastructural examination of central and peripheral components of the neuromuscular system in the SOD1(G93A) mouse and correlated these alterations with early muscle denervation, motor dysfunction, and motoneuron death. We also provide a discussion of published work to review what is known regarding early pathology in the SOD1 mouse model of ALS. The significance of this work is that we have examined early pathology simultaneously in both the spinal cord and peripheral neuromuscular system, and the results are presented in the companion paper (Part II, Results and Discussion). Our results provide evidence as to why a thorough characterization of animal models throughout the life span is critical for a strong foundation to design preclinical trials that may produce meaningful results.

Characterization of early pathogenesis in the SOD1(G93A) mouse model of ALS: part II, results and discussion.

Pathological events are well characterized in amyotrophic lateral sclerosis (ALS) mouse models, but review of the literature fails to identify a specific initiating event that precipitates disease pathology. There is now growing consensus in the field that axon and synapses are first cellular sites of degeneration, but controversy exists over whether axon and synapse loss is initiated autonomously at those sites or by pathology in the cell body, in nonneuronal cells or even in nonmotoneurons (MNs). Previous studies have identified pathological events in the mutant superoxide dismutase 1 (SOD1) models involving spinal cord, peripheral axons, neuromuscular junctions (NMJs), or muscle; however, few studies have systematically examined pathogenesis at multiple sites in the same study. We have performed ultrastructural examination of both central and peripheral components of the neuromuscular system in the SOD1(G93A) mouse model of ALS. Twenty percent of MNs undergo degeneration by P60, but NMJ innervation in fast fatigable muscles is reduced by 40% by P30. Gait alterations and muscle weakness were also found at P30. There was no change in axonal transport prior to initial NMJ denervation. Mitochondrial morphological changes are observed at P7 and become more prominent with disease progression. At P30 there was a significant decrease in excitatory axo-dendritic and axo-somatic synapses with an increase in C-type axo-somatic synapses. Our study examined early pathology in both peripheral and central neuromuscular system. The muscle denervation is associated with functional motor deficits and begins during the first postnatal month in SOD1(G93A) mice. Physiological dysfunction and pathology in the mitochondria of synapses and MN soma and dendrites occur, and disease onset in these animals begins more than 2 months earlier than originally thought. This information may be valuable for designing preclinical trials that are more likely to impact disease onset and progression.

Motoneuron programmed cell death in response to proBDNF.

Motoneurons (MN) as well as most neuronal populations undergo a temporally and spatially specific period of programmed cell death (PCD). Several factors have been considered to regulate the survival of MNs during this period, including availability of muscle-derived trophic support and activity. The possibility that target-derived factors may also negatively regulate MN survival has been considered, but not pursued. Neurotrophin precursors, through their interaction with p75(NTR) and sortilin receptors have been shown to induce cell death during development and following injury in the CNS. In this study, we find that muscle cells produce and secrete proBDNF. ProBDNF through its interaction with p75(NTR) and sortilin, promotes a caspase-dependent death of MNs in culture. We also provide data to suggest that proBDNF regulates MN PCD during development in vivo.

Increased intramuscular nerve branching and inhibition of programmed cell death of chick embryo motoneurons by immunoglobulins from patients with motoneuron disease.

Massive programmed cell death (PCD) of developing chick embryo motoneurons (MNs) occurs in a well defined temporal and spatial sequence between embryonic day (E) 6 and E10. We have found that, when administered in ovo, either circulating immunoglobulins G (IgGs) or cerebrospinal fluid from patients with MN disease can rescue a significant number of chick embryo MNs from normally occurring PCD. An increase of branching of intramuscular nerves was also observed that may account for the rescuing effects of pathologic IgGs. Proteomic analysis and further analysis by ELISA indicated that these effects may be mediated by the interaction of circulating human immunoglobulins with proteins of the semaphorin family.

The rescue of developing avian motoneurons from programmed cell death by a selective inhibitor of the fetal muscle-specific nicotinic acetylcholine receptor.

In an attempt to determine whether the rescue of developing motoneurons (MNS) from programmed cell death (PCD) in the chick embryo following reductions in neuromuscular function involves muscle or neuronal nicotinic acetylcholine receptors (nAChRs), we have employed a novel cone snail toxin alphaA-OIVA that acts selectively to antagonize the embryonic/fetal form of muscle nAChRs. The results demonstrate that alphaA-OIVA is nearly as effective as curare or alpha-bungarotoxin (alpha-BTX) in reducing neuromuscular function and is equally effective in increasing MN survival and intramuscular axon branching. Together with previous reports, we also provide evidence consistent with a transition between the embryonic/fetal form to the adult form of muscle nAChRs in chicken that involves the loss of the gamma subunit in the adult receptor. We conclude that selective inhibition of the embryonic/fetal form of the chicken muscle nAChR is sufficient to rescue MNs from PCD without any involvement of neuronal nAChRs.

Developing postmitotic mammalian neurons in vivo lacking Apaf-1 undergo programmed cell death by a caspase-independent, nonapoptotic pathway involving autophagy.

Previous studies have shown that caspases and Apaf-1 are required for the normal programmed cell death (PCD) in vivo of immature postmitotic neurons and mitotically active neuronal precursor cells. In contrast, caspase activity is not necessary for the normal PCD of more mature postmitotic neurons that are establishing synaptic connections. Although normally these cells use caspases for PCD, in the absence of caspase activity these neurons undergo a distinct nonapoptotic type of degeneration. We examined the survival of these more mature postmitotic neuronal populations in mice in which Apaf-1 has been genetically deleted and find that they exhibit quantitatively normal PCD of developing postmitotic neurons. We next characterized the morphological mode of PCD in these mice and show that the neurons degenerate by a caspase-independent, nonapoptotic pathway that involves autophagy. However, autophagy does not appear to be involved in the normal PCD of postmitotic neurons in which caspases and Apaf-1 are present and functional because quantitatively normal neuronal PCD occurred in the absence of a key gene required for autophagy (ATG7). Finally, we examined the possible role of another caspase-independent type of neuronal PCD involving the apoptosis-inducing factor (AIF). Mice deficient in AIF also exhibit quantitatively normal PCD of postmitotic neurons after caspase inhibition. Together, these data indicate that, when key components of the type 1 apoptotic pathway (i.e., caspases and Apaf-1) are perturbed in vivo, developing postmitotic neurons nonetheless undergo quantitatively normal PCD by a caspase-independent pathway involving autophagy and not requiring AIF.

Exogenous delivery of heat shock protein 70 increases lifespan in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder that results in the progressive loss of motoneurons (MNs) in the CNS. Several survival and death mechanisms of MNs have been characterized and it has been determined that MNs do not appear to mount a complete stress response, as determined by the lack of heat shock protein 70 (Hsp70) upregulation after several stress paradigms. Hsp70 has been shown to confer neuroprotection and the insufficient availability of Hsp70 may contribute to MNs' susceptibility to death in ALS mice. In this study, recombinant human Hsp70 (rhHsp70) was intraperitoneally injected three times weekly, beginning at postnatal day 50 until endstage, to G93A mutant SOD1 (G93A SOD1) mice. The administration of rhHsp70 was effective at increasing lifespan, delaying symptom onset, preserving motor function and prolonging MN survival. Interestingly, injected rhHsp70 localized to skeletal muscle and was not readily detected in the CNS. Treatment with rhHsp70 also resulted in an increased number of innervated neuromuscular junctions compared with control tissue. Together these results suggest rhHsp70 may delay disease progression in the G93A SOD1 mouse via a yet to be identified peripheral mechanism.

The lack of effect of specific overexpression of IGF-1 in the central nervous system or skeletal muscle on pathophysiology in the G93A SOD-1 mouse model of ALS.

The ability of insulin like growth factor 1 (IGF-1) to prevent the pathophysiology associated with amyotrophic lateral sclerosis (ALS) is currently being explored with animal models and in clinical trials with patients. Several studies have reported positive effects of IGF-1 in reducing motor neuron death, delaying the onset of motor performance decline, and increasing life span, in SOD-1 mouse models of ALS and in one clinical trial. However, a second clinical trial produced no positive results raising questions about the therapeutic efficacy of IGF-1. To investigate the effect of specific and sustained IGF-1 expression in skeletal muscle or central nervous system on motor performance, life span, and motor neuron survival, human-IGF-1 transgenic mice were crossed with the G93A SOD-1 mutant model of ALS. No significant differences were found in onset of motor performance decline, life span, or motor neuron survival in the spinal cord, between SOD+/IGF-1+ and SOD+/IGF-1- hybrid mice. IGF-1 concentration levels, measured by radioimmunoassay, were found to be highly increased throughout life in the central nervous system (CNS) and skeletal muscle of IGF-1 transgenic hybrid mice. Additionally, increased CNS weight in SOD+ mice crossbred with CNS IGF-1 transgenic mice demonstrates that IGF-1 overexpression is biologically active even after the disease is fully developed. Taken together, these results raise questions concerning the therapeutic value of IGF-1 and indicate that further studies are needed to examine the relationship between methods of IGF-1 administration and its potential therapeutic value.

Astrocyte and muscle-derived secreted factors differentially regulate motoneuron survival.

During development, motoneurons (MNs) undergo a highly stereotyped, temporally and spatially defined period of programmed cell death (PCD), the result of which is the loss of 40-50% of the original neuronal population. Those MNs that survive are thought to reflect the successful acquisition of limiting amounts of trophic factors from the target. In contrast, maturation of MNs limits the need for target-derived trophic factors, because axotomy of these neurons in adulthood results in minimal neuronal loss. It is unclear whether MNs lose their need for trophic factors altogether or whether, instead, they come to rely on other cell types for nourishment. Astrocytes are known to supply trophic factors to a variety of neuronal populations and thus may nourish MNs in the absence of target-derived factors. We investigated the survival-promoting activities of muscle- and astrocyte-derived secreted factors and found that astrocyte-conditioned media (ACM) was able to save substantially more motoneurons in vitro than muscle-conditioned media (MCM). Our results indicate that both ACM and MCM are significant sources of MN trophic support in vitro and in ovo, but only ACM can rescue MNs after unilateral limb bud removal. Furthermore, we provide evidence suggesting that MCM facilitates the death of a subpopulation of MNs in a p75(NTR) - and caspase-dependent manner; however, maturation in ACM results in MN trophic independence and reduced vulnerability to this negative, pro-apoptotic influence from the target.

Neuromuscular development in the absence of programmed cell death: phenotypic alteration of motoneurons and muscle.

The widespread, massive loss of developing neurons in the central and peripheral nervous system of birds and mammals is generally considered to be an evolutionary adaptation. However, until recently, models for testing both the immediate and long-term consequences of preventing this normal cell loss have not been available. We have taken advantage of several methods for preventing neuronal death in vivo to ask whether rescued neurons [e.g., motoneurons (MNs)] differentiate normally and become functionally incorporated into the nervous system. Although many aspects of MN differentiation occurred normally after the prevention of cell death (including the expression of several motoneuron-specific markers, axon projections into the ventral root and peripheral nerves, ultrastructure, dendritic arborization, and afferent axosomatic synapses), other features of the neuromuscular system (MNs and muscle) were abnormal. The cell bodies and axons of MNs were smaller than normal, many MN axons failed to become myelinated or to form functional synaptic contacts with target muscles, and a subpopulation of rescued cells were transformed from alpha- to gamma-like MNs. Additionally, after the rescue of MNs in myogenin glial cell line-derived neurotrophic factor (MyoGDNF) transgenic mice, myofiber differentiation of extrafusal skeletal muscle was transformed and muscle physiology and motor behaviors were abnormal. In contrast, extrafusal myofiber phenotype, muscle physiology, and (except for muscle strength tests) motor behaviors were all normal after the rescue of MNs by genetic deletion of the proapoptotic gene Bax. However, there was an increase in intrafusal muscle fibers (spindles) in Bax knock-out versus both wild-type and MyoGDNF mice. Together, these data indicate that after the prevention of MN death, the neuromuscular system becomes transformed in novel ways to compensate for the presence of the thousands of excess cells.

Complete dissociation of motor neuron death from motor dysfunction by Bax deletion in a mouse model of ALS.

The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell death-independent role for Bax in facilitating mutant SOD1-mediated motor denervation.

Overexpression of glial cell line-derived neurotrophic factor in the CNS rescues motoneurons from programmed cell death and promotes their long-term survival following axotomy.

To study the role of one of the most potent motoneuron (MN) survival factors, glial cell line-derived neurotrophic factor (GDNF) derived from the CNS, we generated transgenic animals overexpressing GDNF under the control of an astrocyte-specific GFAP promoter. In situ hybridization revealed that GDNF was expressed at high levels in astrocytes throughout the brain and spinal cord. We analyzed the effects of CNS-derived GDNF on MN survival during the period of programmed cell death (PCD) and after nerve axotomy. In GFAP-GDNF mice at E15, E18, and P1, the survival of brachial MNs was increased on average by 30%, lumbar MNs by 20%, and thoracic MNs at P1 by 33%. GDNF also prevented MN PCD in several cranial motor nuclei. We demonstrated for the first time that the number of MNs in the mouse abducens nucleus was also increased by 40%, thus extending known MN populations that are responsive to GDNF. Next, we tested if GDNF could support complete and relatively long-term survival of MNs following neonatal facial nerve axotomy. We found that virtually all MNs (91%) in GFAP-GDNF mice survived for up to 18 weeks post-axotomy. This is the longest GDNF-mediated survival of neonatal MNs reported following axotomy. Most of surviving MNs were not atrophic, and MN-specific ChAT and neurofilament immunoreactivity (IR) were preserved. Furthermore, GDNF attenuated axotomy-induced astroglial activation. These data demonstrate that overexpression of GDNF in the CNS has very profound effects on MN survival both during the PCD period and after neuronal injury. GFAP-GDNF mice will be valuable to study the effects of CNS-derived GDNF in mouse models of MN degenerative diseases and axonal regeneration in vivo.

Cardiotrophin-like cytokine/cytokine-like factor 1 is an essential trophic factor for lumbar and facial motoneurons in vivo.

The ciliary neurotrophic factor alpha-receptor (CNTFRalpha) is required for motoneuron survival during development, but the relevant ligand(s) has not been determined. One candidate is the heterodimer formed by cardiotrophin-like cytokine (CLC) and cytokine-like factor 1 (CLF). CLC/CLF binds to CNTFRalpha and enhances the survival of developing motoneurons in vitro; whether this novel trophic factor plays a role in neural development in vivo has not been tested. We examined motor and sensory neurons in embryonic chicks treated with CLC and in mice with a targeted deletion of the clf gene. Treatment with CLC increased the number of lumbar spinal cord motoneurons that survived the cell death period in chicks. However, this effect was regionally specific, because brachial and thoracic motoneurons were unaffected. Similarly, newborn clf-/- mice exhibited a significant reduction in lumbar motoneurons, with no change in the brachial or thoracic cord. Clf deletion also affected brainstem motor nuclei in a regionally specific manner; the number of motoneurons in the facial but not hypoglossal nucleus was significantly reduced. Sensory neurons of the dorsal root ganglia were not affected by either CLC treatment or clf gene deletion. Finally, mRNA for both clc and clf was found in skeletal muscle fibers of embryonic mice during the motoneuron cell death period. These findings support the view that CLC/CLF is a target-derived factor required for the survival of specific pools of motoneurons. The in vivo actions of CLC and CLF can account for many of the effects of CNTFRalpha on developing motoneurons.

Cell cycle events distinguish sensory neuronal death from motoneuron death as a result of trophic factor deprivation.

The clarification of mechanisms of developmental cell death may provide hints in the prevention of pathological neuronal death. The execution phase of cell death has been extensively characterized; however, events that occur prior to this phase are less well understood. Previous studies have suggested that terminally differentiated neurons induced to die in various experimental paradigms may be making an abortive attempt to reenter the cell cycle. We have examined this process in postmitotic motoneurons and dorsal root ganglia sensory neurons in the developing chick embryo in vitro and in vivo. An examination of the programmed cell death of postmitotic motoneurons does not implicate a role for cell cycle-related proteins. We did, however, observe a decrease in the amount of cell death in dorsal root ganglion cells of embryos treated with cell cycle inhibitors. These results indicate that upstream initiators of the neuronal cell death pathway vary between phenotypes.

GDNF and BDNF alter the expression of neuronal NOS, c-Jun, and p75 and prevent motoneuron death following spinal root avulsion in adult rats.

In the present study, we examined the effects of glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and insulin growth factor (IGF-1) on adult motoneuron survival following spinal root avulsion. The expression of neuronal nitric oxide synthase (nNOS), c-Jun, and the low-affinity neurotrophin receptor (P75) following treatment with these neurotrophic factors was also examined. In control animals, approximately 80% of spinal motoneurons were nNOS positive at 3 weeks following the lesion, whereas in GDNF or BDNF treated animals no nNOS positive motoneurons were found at the same time point. Following injury and treatment with GDNF and BDNF increased numbers of motoneurons were c-Jun and P75 positive. By 6 weeks following the lesion, only approximately 28% of motoneurons persisted in control animals whereas about 90% of motoneurons survived injury following treatment with either GDNF or BDNF. In contrast, CNTF and IGF-1 were ineffective in either inhibiting nNOS expression or preventing motoneuron death. Our results provide in vivo evidence that the survival of injured adult mammalian motoneurons can be promoted by specific neurotrophic factors, and that this effect is associated with inhibition of nNOS expression and up-regulation of c-Jun and P75 expression.

Neuromuscular development after the prevention of naturally occurring neuronal death by Bax deletion.

The removal of excess neurons by programmed cell death (PCD) is believed to be critical for the proper development and function of the nervous system. A major role of this neuronal loss is to attain quantitative matching of neurons with their targets and afferents. Because motoneurons (MNs) in Bax knock-out (Bax KO) mice fail to undergo PCD in the face of normal target muscle development, we asked whether the excess rescued neurons in Bax KO mice can develop normally. We observed many small atrophied MNs in postnatal Bax KO mice, and these failed to innervate limb muscle targets. When examined embryonically during the PCD period, however, these excess MNs had initiated target innervation. To examine whether a limitation in trophic factor availability is responsible for postnatal MN atrophy and loss of innervation, we applied glial cell line-derived neurotrophic factor (GDNF) to neonatal mice. GDNF injection for 7-14 d induced the regrowth and reinnervation of muscle targets by atrophic MNs in Bax KO mice and prevented the normal postnatal death of MNs in wild-type mice. These results indicate that, although initially all of the MNs, including those rescued by Bax deletion, are able to project to and innervate targets, because of limited target-derived signals required for maintaining innervation and growth, only a subpopulation can grow and retain target contacts postnatally. Although sensory neurons in the dorsal root ganglia are also rescued from PCD by Bax deletion, their subsequent development is less affected than that of MNs.

Rescue of developing spinal motoneurons from programmed cell death by the GABA(A) agonist muscimol acts by blockade of neuromuscular activity and increased intramuscular nerve branching.

Blockade of neuromuscular activity in the chick embryo during the period of programmed cell death of motoneurons results in a complete rescue of these cells. Understanding the cellular mechanisms that mediate this counterintuitive effect is of considerable interest with respect to the regulation of motoneuron survival during development as well as for understanding why motoneurons die pathologically. Although considerable evidence supports the role of a peripheral site of action at the neuromuscular junction in mediating the rescue of motoneurons following activity blockade, some evidence also supports a role for central nervous system (CNS) neurons. For example, the rescue of motoneurons by curare has been reported to be blocked by the GABA(A) agonist muscimol via its actions on CNS neurons. We have carried out a series of studies to further investigate this interesting observation. Surprisingly, we find that: (1) muscimol blocks activity and rescues MNs in a dose-dependent manner, similar to curare; (2) muscimol's effects on MN survival appear to be mediated by its action on intramuscular nerve branching, similar to curare; and (3) although muscimol acts centrally, the effects of muscimol on MN survival and axon branching are mediated peripherally at the neuromuscular junction, similar to curare. Because muscimol reduces MN depolarization these data also suggest that the depolarization of MNs by afferents is not required for promoting MN survival. Taken together, these data provide further evidence in support of a peripheral site of action of activity blockade in rescuing motoneurons from developmental cell death.

Developmental expression of nicotinic receptors in the chick and human spinal cord.

Naturally occurring programmed cell death of lumbar motor neurons in the chick spinal cord occurs between embryonic day (E) 6 and E12; whereas, a peak of motor neuron degeneration in the human spinal cord occurs between 12 and 16 weeks gestation. One of the major neurotransmitters, acetylcholine, is released from the embryonic motor neuron early in development and is thought to be responsible for early muscle activity that serves as a signal for regulating motor neuron survival. The effects of acetylcholine are mediated by two functionally distinct classes of receptors; namely, muscarinic and nicotinic with nicotinic receptors being used at the neuromuscular synapse. In this study, we determined the developmental expression profile of nicotinic acetylcholine receptor subunits in the chick and human lumbar motor neurons and skeletal muscle using reverse transcription polymerase chain reaction, immunoblots, and immunocytochemistry. Our results show that, in the chick, nicotinic receptor subunits alpha1, alpha4, alpha7, alpha8, and beta2 appear to be regulated during the process of naturally occurring motor neuron cell death in the spinal cord. A new finding was the expression of alpha8 mRNA and protein from E4.5 through E7 in chick motor neurons. Interestingly, we also found that, at E14, alpha8 protein was localized only in sensory dorsal horn neurons. In the developing human spinal cord, we determined that nicotinic receptor subunits alpha1, alpha2, alpha3, alpha4, alpha7, beta2, and beta3 were expressed before the programmed cell death period, and alpha2, alpha4, alpha7, beta2, beta3, and beta4 were expressed during the programmed cell death period. Our data demonstrate that neuronal and muscle nicotinic receptor mRNAs and proteins are expressed during important embryonic periods. This finding raises the possibility that nicotinic receptors play an important role in the spinal cord and skeletal muscle during early development.

In vivo analysis of Schwann cell programmed cell death in the embryonic chick: regulation by axons and glial growth factor.

The present study uses the embryonic chick to examine in vivo the mechanisms and regulation of Schwann cell programmed cell death (PCD) in spinal and cranial peripheral nerves. Schwann cells are highly dependent on the presence of axons for survival because the in ovo administration of NMDA, which excitotoxically eliminates motoneurons and their axons by necrosis, results in a significant increase in apoptotic Schwann cell death. Additionally, pharmacological and surgical manipulation of axon numbers also affects the relative amounts of Schwann cell PCD. Schwann cells undergoing both normal and induced PCD display an apoptotic-like cell death, using a caspase-dependent pathway. Furthermore, axon elimination results in upregulation of the p75 and platelet-derived growth factor receptors in mature Schwann cells within the degenerating ventral root. During early development, Schwann cells are also dependent on axon-derived mitogens; the loss of axons results in a decrease in Schwann cell proliferation. Axon removal during late embryonic stages, however, elicits an increase in proliferation, as is expected from these more differentiated Schwann cells. In rodents, Schwann cell survival is regulated by glial growth factor (GGF), a member of the neuregulin family of growth factors. GGF administration to chick embryos selectively rescued Schwann cells during both normal PCD and after the loss of axons, whereas other trophic factors tested had no effect on Schwann cell survival. In conclusion, avian Schwann cells exhibit many similarities to mammalian Schwann cells in terms of their dependence on axon-derived signals during early and later stages of development.