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.

Development of microglia in the chick embryo spinal cord: implications in the regulation of motoneuronal survival and death.

The role of microglia during normal development of the nervous system is still not well understood. In the present study, a chick embryo model was used to examine the development of microglia in the spinal cord and characterize their changes in response to naturally occurring and pathological death of motoneurons (MNs). The microglial response to MN axotomy and the effects of microglial activation on MN survival were also studied. We found that: 1) macrophages/microglial cells were present in the spinal cord at early developmental stages (E3) and that they were recruited after normal and induced MN apoptosis; 2) although many microglial cells were seen phagocytosing apoptotic bodies, a proportion of dying cells were devoid of engulfing microglia; 3) axotomy of mature MNs was accompanied by microglial activation in the absence of MN death; 4) excitotoxic (necrotic) MN death provoked a rapid and massive microglial recruitment with phagocytic activity; 5) lipopolysaccharide-induced microglial activation in vivo resulted in the death of immature, but not mature, microglia; and 6) overactivation of microglia modulated the survival of mature MNs, either by killing them or by enhancing their vulnerability to die in response to a mild injury. Taken together, these observations indicate that normal microglia do not play an active role in triggering apoptosis of developing MNs. Rather, they act as phagocytes for the removal of dying cells during the process of programmed cell death.

Excitotoxic motoneuron disease in chick embryo evolves with autophagic neurodegeneration and deregulation of neuromuscular innervation.

In the chick embryo, in ovo application of NMDA from embryonic day (E) 5 to E9 results in selective damage to spinal cord motoneurons (MNs) that undergo a long-lasting degenerative process without immediate cell death. This contrasts with a single application of NMDA on E8, or later, which induces massive necrosis of the whole spinal cord. Chronic MN degeneration after NMDA implies transient incompetence to develop programmed cell death, altered protein processing within secretory pathways, and late activation of autophagy. Chronic NMDA treatment also results in an enlargement of thapsigargin-sensitive Ca(2+) stores. In particular MN pools, such as sartorius-innervating MNs, the neuropeptide CGRP is accumulated in somas, peripheral axons and neuromuscular junctions after chronic NMDA treatment, but not in embryos paralyzed by chronic administration of curare. Intramuscular axonal branching is also altered severely after NMDA: it usually increases, but in some cases a marked reduction can also be observed. Moreover, innervated muscle postsynaptic sites increase by NMDA, but to a lesser extent than by curare. Because some of these results show interesting homologies with MN pathology in human sporadic ALS, the model presented here provides a valuable tool for advancing in the understanding of some cellular and molecular processes particularly involved in this disease.

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.