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.     

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.     

Calpain Inhibition Protects Spinal Motoneurons from the Excitotoxic Effects of AMPA In vivo

Microdialysis perfusion of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in rat lumbar spinal cord produces severe motoneuron damage and consequently hindlimb paralysis. Here we studied the time course of the AMPA-induced neurodegenerative changes and motor alterations, and the protective effect of leupeptin, an inhibitor of calpain, a Ca(2+)-activated protease. Paralysis occurs at 4-6 h after AMPA perfusion, but cresyl violet staining showed that motoneuron damage starts at about 3 h and progresses until reaching 50% neuronal loss at 6 h and 90% loss at 12 h. In contrast, choline acetyltransferase (ChAT) immunohistochemistry revealed that the enzyme is already decreased at 30 min after AMPA perfusion and practically disappears at 3 h. Microdialysis coperfusion of leupeptin with AMPA prevented the motor alterations and paralysis and remarkably reduced both the decrement in ChAT immunoreactivity and the loss of motoneurons. We conclude that an increased Ca(2+) influx through Ca(2+)-permeable AMPA receptors activates calpain, and as a consequence ChAT content decreases earlier than other Ca(2+)-dependent processes, including the proteolytic activity of calpain, cause the death of motoneurons.     

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.     

Biological studies of a putative avian muscle-derived neurotrophic factor that prevents naturally occurring motoneuron death in vivo

A series of in vivo studies have been carried out using the chick embryo to address several critical questions concerning the biological, and to a lesser extent, the biochemical characteristics of a putative avian muscle-derived trophic agent that promotes motoneuron survival in vivo. A partially purified fraction of muscle extract was shown to be heat and trypsin sensitive and rescued motoneurons from naturally occurring cell death in a dose-dependent fashion. Muscle extract had no effect on mitotic activity in the spinal cord and did not alter cell number when administered either before or after the normal cell death period. The survival promoting activity in the muscle extract appears to be developmentally regulated. Treatment with muscle extract during the cell death period did not permanently rescue motoneurons. The motoneuron survival-promoting activity found in skeletal muscle was not present in extracts from a variety of other tissues, including liver, kidney, lung, heart, and smooth muscle. Survival activity was also found in extracts from fetal mouse, rat, and human skeletal muscle. Conditioned medium derived from avian myotube cultures also prevented motoneuron death when administered in vivo to chick embryos. Treatment of embryos in ovo with muscle extract had no effect on several properties of developing muscles. With the exception of cranial motoneurons, treatment with muscle extract did not promote the survival of several other populations of neurons in the central and peripheral nervous system that also exhibit naturally occurring cell death. Initial biochemical characterization suggests that the activity in skeletal muscle is an acidic protein between 10 and 30 kD. Examination of a number of previously characterized growth and trophic agents in our in vivo assay have identified several molecules that promote motoneuron survival to one degree or another. These include S100β, brain-derived neurotrophic factor (BDNF), neurotrophin 4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), transforming growth factor β (TGFβ), platelet-derived growth factor-AB (PDGF-AB), leukemia inhibitory factor (CDF/LIF), and insulin-like growth factors I and II (IGF). By contrast, the following agents were ineffective: nerve growth factor (NGF), neurotrophin-3 (NT3), epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF, bFGF), and the heparin-binding growth-associated molecule (HB-GAM). Of those agents that were effective, CDF/LIF, IGF-1 and -2, BDNF, and TGF are reported to be expressed in developing or adult muscle. Studies are underway to determine whether the survival activity found in avian muscle extract can be accounted for by one or more of these growth factors. Of all the tissue extracts and purified proteins tested here, only the neurotrophins—NGF, NT-3, and BDNF (but not NT-4/5)—rescured sensory neurons from naturally occurring cell death. © 1993 John Wiley & Sons, Inc.     

Relevance of motoneuron specification and programmed cell death in embryos to therapy of ALS

The molecular cues that generate spinal motoneurons in early embryonic development are well defined. Motoneurons are generated in excess and consequently undergo a natural period of programmed cell death. Although it is not known exactly how motoneurons compete for survival in embryonic development, it is hypothesized that they rely on the ability to access limited amounts of trophic factors from peripheral tissues, a process that is tightly regulated by skeletal muscle activity. Attempts to elucidate the molecular mechanisms that underlie motoneuron generation and programmed cell death in embryos have led to various effective strategies for treating injury and disease in animal models. Such studies provide great hope for the amelioration of human amyotrophic lateral sclerosis (ALS), a devastating progressive motoneuron degenerative disease. Here we review the clinical relevance of studying motoneuron specification and death during embryonic development.     

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.     

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.     

Motoneuronal death during spinal cord development is mediated by oxidative stress

The involvement of reactive oxygen species (ROS) in neuronal death has been determined in culture, and in association with several neurodegenerative disorders. We examined whether ROS participate in the cell death observed during spinal cord development. We found that the general pattern of high ROS levels, gene expression for some antioxidant enzymes, and motoneuron death correlated positively along spinal cord development. ROS were reduced in spinal cords cultured in the presence of a synthetic superoxide dismutase and catalase mimetic, with a concomitant reduction in cell death and an increase in the number of motoneurons. The number of motoneurons was higher in spinal cords treated with the antioxidant than in those treated with caspase inhibitors. In general, the increase in motoneuron survival did not correlate with the reduction in cells undergoing DNA degradation in the motoneuronal region. These results suggest that ROS are signaling molecules controlling caspase-dependent and caspase-independent programmed motoneuron death, and support the hypothesis that this mechanism is abnormally turned on in some neurodegenerative disorders and aging.     

The development of motoneurons in the embryonic spinal cord of the mouse mutant, muscular dysgenesis (mdg/mdg): survival, morphology, and biochemical differentiation

Motoneuron development was studied in the spinal cord of the mouse mutant, muscular dysgenesis, between embryonic days (E) 13 and 18. Dysgenic embryos are characterized by the absence of neuromuscular activity (motility) and exhibit a number of other striking changes in neuromuscular development. Many of these changes have also been observed in chick embryos chronically treated with neuromuscular blocking agents that suppress motility. Motoneuron survival, as well as several other aspects of neuronal development, was examined in the thoracic and lumbar spinal cords of mutant and control embryos. There was a significant decrease in motoneuron numbers in control embryos indicating the presence of naturally occurring cell death in the mouse spinal cord. At all ages examined, the dysgenic embryos had significantly more healthy and significantly fewer degenerating motoneurons than controls. There were no differences in the number of dorsal root ganglion neurons or in any of the other morphometric parameters examined between mutant and control embryos. Creatine kinase activity, a marker for myofiber maturation, was significantly reduced in the limb musculature of mutant embryos. Choline acetyltransferase activity was significantly increased in the spinal cord of mutant embryos. No significant differences were observed in spinal cord levels of acetylcholinesterase activity between control and mutant embryos. The absence of muscle contractions in the dysgenic mouse is associated with a number of changes in neuromuscular development, including a substantial reduction of naturally occurring motoneuron death.     

Cyclin dependent kinase inhibitors prevent apoptosis of postmitotic mouse motoneurons

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle which is characterized by increased expression of cyclins, such as cyclin D1, prior to death. However, such cyclins activation prior to apoptotic cell death remains controversial. Many neurological disorders are characterized by neuronal loss, particularly amyotrophic lateral sclerosis (ALS). ALS is a motoneuronal degenerative condition in which motoneuron loss could be due to an inappropriate return of these cells in the cell cycle. In the present study, we observed that deprivation of neurotrophic factor in purified motoneuron cultures induces an apoptotic pathway. After neurotrophic factor withdrawal, DAPI (4,6-diamidin-2-phenylindol dichlorohydrate) staining revealed the presence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic body. Similarly, release of apoptotic microparticles and activation of caspases-3 and -9 were observed within the first hours following neurotrophic factor withdrawal. Next, we tested whether inhibition of cell cycle-related cyclin-dependent kinases (cdks) can prevent motoneuronal cell death. We showed that three cdk inhibitors, olomoucine, roscovitine and flavopiridol, suppress the death of motoneurons. Finally, we observed early increases in cyclin D1 and cyclin E expression after withdrawal of neurotrophic factors. These findings support the hypothesis that after removal of trophic support, post-mitotic neuronal cells die due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner.     

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.     

Skeletal Muscle Proteins Stimulate Cholinergic Differentiation of Human Neuroblastoma Cells

Extracts of rat skeletal muscle contain substances that enhance the development of choline acetyltransferase (ChAT) in the cholinergic human neuroblastoma cell line LA-N-2. The ChAT enhancing activity in muscle extract was purified to homogeneity by preparative gel electrophoresis and reverse-phase HPLC. The active factor is biochemically and immunologically identical to ChAT development factor, (CDF), the skeletal muscle factor that enhances ChAT activity in enriched cultures of embryonic rat motoneurons and rescues motoneurons from naturally occurring cell death in vivo. CDF increases the specific ChAT activity of LA-N-2 cells fivefold after 6 days in culture, but does not affect their growth or metabolic activity. Basic fibroblast growth factor also increases ChAT activity in LA-N-2 cells and its effect is additive with that of CDF. In contrast, neither insulin-like growth factor-1, epidermal growth factor, nor nerve growth factor affected the ChAT activity of LA-N-2 cells. Our study demonstrates for the first time that CDF can directly affect the development of neuronal properties in a homogeneous population of cells, and that the effects of CDF are separate from those of other types of trophic factors.     

Mechanisms of insulin-like growth factor regulation of programmed cell death of developing avian motoneurons

During development of the avian neuromuscular system, lumbar spinal motoneurons (MNs) innervate their muscle targets in the hindlimb coincident with the onset and progression of MN programmed cell death (PCD). Paralysis (activity blockade) of embryos during this period rescues large numbers of MNs from PCD. Because activity blockade also results in enhanced axonal branching and increased numbers of neuromuscular synapses, it has been postulated that following activity blockade, increased numbers of MNs can gain access to muscle-derived trophic agents that prevent PCD. An assumption of the access hypothesis of MN PCD is the presence of an activity-dependent, muscle-derived sprouting or branching agent. Several previous studies of sprouting in the rodent neuromuscular system indicate that insulin-like growth factors (IGFs) are candidates for such a sprouting factor. Accordingly, in the present study we have begun to test whether the IGFs may play a similar role in the developing avian neuromuscular system. Evidence in support of this idea includes the following: (a) IGFs promote MN survival in vivo but not in vitro; (b) neutralizing antibodies against IGFs reduce MN survival in vivo; (c) both in vitro and in vivo, IGFs increase neurite growth, branching, and synapse formation; (d) activity blockade increases the expression of IGF-1 and IGF-2 mRNA in skeletal muscles in vivo; (e) in vivo treatment of paralyzed embryos with IGF binding proteins (IGF-BPs) that interfere with the actions of endogenous IGFs reduce MN survival, axon branching, and synapse formation; (f) treatment of control embryos in vivo with IGF-BPs also reduces synapse formation; and (g) treatment with IGF-1 prior to the major period of cell death (i.e., on embryonic day 6) increases subsequent synapse formation and MN survival and potentiates the survival-promoting actions of brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF) administered during the subsequent 4- to 5-day period of PCD. Collectively, these data provide new evidence consistent with the role of the IGFs as activity-dependent, muscle-derived agents that play a role in regulating MN survival in the avian embryo. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 379–394, 1998     

Ethanol influences on the chick embryo spinal cord motor system: Analyses of motoneuron cell death,motility, and target trophic factor activity and in vitro analyses of neurotoxicity and trophic factor neuroprotection

A series of in vivo and in vitro experiments were conducted to determine the influence of prenatally administered ethanol on several aspects of the developing chick embryo spinal cord motor system. Specifically, we examined: (1) the effect of chronic ethanol administration during the natural cell death period on spinal cord motoneuron numbers; (2) the influence of ethanol on ongoing embryonic motility; (3) the effect of ethanol exposure on neurotrophic activity in motoneuron target tissue (limbbud); and (4) the responsiveness of cultured spinal cord neurons to ethanol, and the potential of target-derived neurotrophic factors to ameliorate ethanol neurotoxicity. These studies revealed the following: Chronic prenatal ethanol exposure reduces the number of motoneurons present in the lateral motor column after the cell death period [embryonic day 12 (E12)]. Ethanol tends to inhibit embryonic motility, particularly during the later stages viewed (E9-E11). Chronic ethanol exposure reduces the neurotrophic activity contained in target muscle tissue. Such diminished support could contribute to the observed motoneuron loss. Direct exposure of spinal cord neurons to ethanol decreases neuronal survival and process outgrowth in a dose-dependent manner, but the addition of target muscle extract to ethanol-containing cultures can ameliorate this ethanol neurotoxicity. These studies demonstrate ethanol toxicity in a population not previously viewed in this regard and suggest a mechanism that may be related to this cell loss (i.e., decreased neurotrophic support). © 1995 John Wiley & Sons, Inc.     

Differential sensitivity of cholinergic and GABAergic neurons in chick embryos treated intracerebrally with ethanol at 8 days of embryonic age

We have shown that in embryos treated with ethanol in ovo during days 1–3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferse (ChAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in both cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergic neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick.     

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.