Period Of Developmental Cell Death Research Articles

The Timing of Activity Is a Regulatory Signal During Development of Neural Connections

In PNS and CNS remarkable rearrangements occur soon after the connections are laid down in the course of embryonic life. These processes clearly follow the period of developmental cell death and mostly take place during the very beginning of postnatal life. They consist in changes of the peripheral fields of neurons, marked by elimination of many inputs, while others undergo further maturation and strengthening. Along the efforts to uncover the signals that regulate development, it turned out that while the initial construction of the circuits is heavily based on chemical cues, the subsequent rearrangement is markedly influence by activity. Here we describe experiments testing the influence on developmental plasticity of a particular aspect of activity, the timing of nerve impulses in the competing inputs. Two recent investigations are reviewed, indicating strikingly similar developmental features in quite different systems, neuromuscular and visual. A sharp contrast between the effects of synchrony and asynchrony emerges, indicating that Hebb-related activity rules are important not only for learning but also for development.

Long-Distance Control of Synapse Assembly by Target-Derived NGF

We report a role for long-distance retrograde neurotrophin signaling in the establishment of synapses in the sympathetic nervous system. Target-derived NGF is both necessary and sufficient for formation of postsynaptic specializations on dendrites of sympathetic neurons. This, in turn, is a prerequisite for formation of presynaptic specializations, but not preganglionic axonal ingrowth from the spinal cord into sympathetic ganglia. We also find that NGF-TrkA signaling endosomes travel from distal axons to cell bodies and dendrites where they promote PSD clustering. Furthermore, the p75 neurotrophin receptor restricts PSD formation, suggesting an important role for antagonistic NGF-TrkA and p75 signaling pathways during retrograde control of synapse establishment. Thus, in addition to defining the appropriate number of sympathetic neurons that survive the period of developmental cell death, target-derived NGF also exerts control over the degree of connectivity between the spinal cord and sympathetic ganglia through retrograde control of synapse assembly.

TIEG1 facilitates transforming growth factor-beta-mediated apoptosis in the oligodendroglial cell line OLI-neu.

Transforming growth factor-beta (TGF-beta) plays an important role during the period of developmental cell death in the nervous system. Using the oligodendroglial precursor cell line OLI-neu, we have previously established an in vitro system to analyze TGF-beta-mediated cell death on the molecular level. We could show that the Krüppel-like Zn-finger transcription factor TIEG1 was up-regulated after TGF-beta stimulation of OLI-neu cells and mimicked TGF-beta effects in these cells; i.e., overexpression of TIEG1 in OLI-neu cells induced apoptosis as shown by apoptosis ELISA, DNA fragmentation, and caspases-3 activation. The apoptotic pathway seemed to be initiated by repressing the expression of the antiapoptotic protein Bcl-XL. In contrast, the reporter activity of a SMAD consensus promoter was induced, whereas the promoter activity of the inhibitory SMAD7 was reduced, suggesting that SMAD-dependent TGF-beta responses, such as TGF-beta-induced apoptosis, are enhanced in the presence of TIEG1.

Transforming growth factor-beta and tumor necrosis factor-alpha cooperate to induce apoptosis in the oligodendroglial cell line OLI-neu.

As shown previously, transforming growth factor-beta (TGF-beta) plays an important role during the period of developmental cell death in the nervous system. As with neurons, oligodendrocytes are generated in excess and eliminated by apoptosis. The present study was aimed at investigating the possible interaction of TGF-beta with tumor necrosis factor-alpha (TNF-alpha) in the regulation of cell death in oligodendroglial precursor cells and analyzing the underlying signaling mechanisms. We show that both factors induce apoptosis independently, but cooperate when applied together. The investigation of the signaling events revealed an important role of the JNK pathway during induction of apoptosis. TGF-beta seemed to be more efficient at inducing a release in cytochrome c from mitochondria than TNF-alpha. This might be the consequence of decreased Bcl-xL levels observed in cells treated with TGF-beta but not with TNF-alpha. Both factors stimulated caspase-3 activity, which could be inhibited by caspase-8 or caspase-9 inhibitors. Therefore, we conclude that TNF-alpha and TGF-beta affect partially common pathways but also regulate different steps in the apoptotic cascade.

TGF-beta induces cell death in the oligodendroglial cell line OLI-neu.

We have shown that TGF-beta plays an important role during the period of developmental cell death in the nervous system. Immunoneutralization of TGF-beta prevents ontogenetic neuron death in vivo. Like neurons, oligodendrocytes are generated in excess and eliminated by apoptosis. It has been shown that oligodendrocyte progenitors and newly formed oligodendrocytes are especially susceptible to apoptosis. We choose the oligodendrocyte precursor cell line OLI-neu to address the question if TGF-beta could play a role for the control of oligodendrocyte proliferation and cell death. Flow cytometric analysis revealed that OLI-neu cells arrested in the G1 phase of the cell cycle underwent apoptosis in response to TGF-beta. TUNEL assays, apoptosis ELISA, and caspase assays substantiated the finding that OLI-neu cells died after TGF-beta treatment. Cell death could be inhibited by application of pan-caspase or caspase 8 and 9 inhibitors, whereas the inhibition of calpain was unaffected. Furthermore, we found a reduction of bcl-X(L) at the protein as well as at the mRNA level, while p27 was upregulated. The Smad cascade was activated while TGF-beta reduced the activity of the p42/p44 MAP kinase pathway. Together, these data show that TGF-beta induced apoptotic cell death in cells of oligodendroglial origin, whereby the signaling cascade involved the downregulation of antiapoptotic signaling such as bcl-X(L) leading to the activation of caspases.

Characterization of dopaminergic midbrain neurons in a DBH:BDNF transgenic mouse.

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in the survival and differentiation of central nervous system neurons, including dopaminergic cells in culture. To determine whether BDNF might play a role in the development of dopaminergic neurons in vivo, we used a previously characterized transgenic mouse (DBH:BDNF) that overexpresses BDNF in adrenergic and noradrenergic neurons as a result of fusion of the BDNF gene to the dopamine beta-hydroxylase (DBH) gene promoter. We quantified dopaminergic neuronal profiles at four midbrain coronal levels and compared DBH:BDNF transgenic animals with wild-type mice of the same genetic background. Analysis of sections immunostained with tyrosine hydroxylase (TH) showed that the mean number of dopaminergic neurons in the four selected midbrain sections was 52% greater (one-way analysis of variance, P < 0.0005) in transgenic mice (2,165 +/- 55 S. E.M., n = 4) than in control mice (1,428 +/- 71 S.E.M., n = 4). The increase in dopaminergic neuron profile count in DBH:BDNF transgenic animals was confirmed by analysis of the pars compacta of the substantia nigra on Nissl-stained sections. Surface area of the reference region of interest containing TH-immunoreactive neurons was similar in transgenic and control mice. Regional analysis of different midbrain areas containing dopaminergic neurons suggested that the increase in cell profile count occurs in a relatively homogeneous manner. Comparison of TH-immunoreactive cell size showed a tendency for smaller neurons in transgenic animals, but the difference was not statistically significant. We conclude that DBH:BDNF transgenic mice show increased number of TH-immunoreactive cells in the midbrain. We propose that BDNF rescues dopaminergic neurons from the perinatal period of developmental cell death as a consequence of increased anterograde transport of the neurotrophin via the coeruleonigral projection.

Trophic effects of androgen: Development and hormonal regulation of neuron number in a sexually dimorphic vocal motor nucleus

In Xenopus laevis, the laryngeal motor nucleus (n. of cranial nerves IX-X) is part of a sexually differentiated, androgen sensitive neuromuscular system devoted to vocalization. Adult males have more n. IX-X neurons than females; however, during development of n. IX-X, the rate of neurogenesis does not appear to differ between the sexes. In this study, we explored the role of naturally occurring cell death in the development of this nucleus and asked whether cell death might be involved in establishing the sex difference in neuron number. Counts of n. IX-X neurons reveal that at tadpole stage 56, males and females have similar numbers of n. IX-X neurons, but by stage 64 male neuron numbers are greater. This sex difference arises owing to a greater net loss of neurons in females-males lose approximately 25% of their n. IX-X neurons between stages 56 and 64, while females lose approximately 47%. Sexual differentiation of n. IX-X neuron number coincides with a period of developmental cell death, as evidenced by terminal transferase-mediated dUTP nick-end labeling and the presence of pyknotic nuclei in n. IX-X. A role for gonadal hormones in controlling cell number was examined by treating tadpoles with exogenous androgen and determining the number of n. IX-X neurons at stage 64. Dihydrotestosterone (DHT) treatment from the beginning of the cell death period (stage 54) until stage 64 had no effect on the number of n. IX-X neurons in males but did significantly increase n. IX-X neuron number in females. This increase was sufficient to abolish the sex difference normally observed at stage 64. Although DHT induced increases in female neuron number, it did not induce increases in cell proliferation or addition of newly born neurons to n. IX-X. DHT may therefore have increased neuron number by protecting cells from death. We conclude that androgens can influence the survival of n. IX-X neurons during a period of naturally occurring cell death, and that this action of androgen is critical to the development of sex differences in n. IX-X neuron number.

Bax promotes neuronal cell death and is downregulated during the development of the nervous system

The Bcl-2 and Bcl-x proteins suppress programmed cell death, whereas Bax promotes apoptosis. We investigated the pattern of expression of Bcl-2, Bax and Bcl-x during neuronal differentiation and development. All three proteins were widely expressed in neonatal rats but, in the adult, Bax levels were 20- to 140-fold lower in the cerebral cortex, cerebellum and heart muscle, whereas Bcl-x was not downregulated in any of the tissues examined. In the cerebral cortex and cerebellum, the decrease in Bax levels occurred after the period of developmental cell death. Further, microinjection of a Bax expression vector into cultured sympathetic neurons, which depend on nerve growth factor for survival, induced apoptosis in the presence of survival factor and increased the rate of cell death after nerve growth factor withdrawal. This effect could be blocked by co-injection of an expression vector for Bcl-xL or for the baculovirus p35 protein, an inhibitor of caspases (ICE-like proteases). These results suggest that, during development, the sensitivity of neurons to signals that induce apoptosis may be regulated by modulating Bax levels and that Bax-induced death requires caspase activity.

Members of several gene families influence survival of rat motoneurons in vitro and in vivo.

The survival and functional maintenance of spinal motoneurons, both during the period of developmental cell death and in adulthood, have been shown to be dependent on trophic factors. In vitro experiments have previously been used to identify several survival factors for motoneurons, including CNTF, LIF, and members of the neurotrophin, FGF, and IGF gene families. Some of these factors have also been shown to be active in vivo, either on chick motoneurons during embryonic development or on lesioned facial and spinal motoneurons of the newborn rat. Here we demonstrate that lesioned newborn rat facial motoneurons can be rescued by NT-4/5, IGF-I, and LIF. Furthermore, in contrast to chick motoneurons, the survival of isolated embryonic rat motoneurons can be maintained by the neurotrophins BDNF, NT-3, and NT-4/5. IGF-I and FGF-5 were also active in this system, each supporting more than 50% of the originally plated neurons. The responsiveness of motoneurons to multiple factors in vitro and in vivo suggests that motoneuron survival and function are regulated by the coordinated actions of members of different gene families.

Altered protein binding to the octamer motif appears to be an early event in programmed neuronal cell death.

Electrophoretic mobility-shift assays were used to characterize binding of nuclear proteins to consensus sequences for Sp1, E2F, octamer, and cAMP responsive enhancer element (CRE) during neuronal death in vitro after removal of nerve growth factor (NGF). Molecular events occurring prior to cell death in terminally differentiated PC12 cells could be divided into three phases: (i) within 2 hr of removing NGF, binding to the octamer sequence decreased, (ii) after 5-7 hr an increase in binding to CRE occurred; and (iii) after 14 hr (the point at which 50% of the cells are committed to die) a decrease in binding to the Sp1 sequence occurred. Assays performed with extracts from sympathetic ganglia indicated that changes in binding to CRE and octamer motifs also occurred during the period of developmental cell death in vivo. Double-stranded oligonucleotides were delivered to neurons to act as dominant negative "promoters" unable to couple to transcriptional events but capable of binding and sequestering transcription factors. Double-stranded but not single-stranded octamer oligonucleotides increased cell death of primary cultures of sympathetic neurons. Most of the induced neuronal cell death could be blocked with NGF, which is consistent with oligonucleotides activating an endogenous death program rather than having a nonspecific toxic effect. Other double-stranded oligonucleotides as well as a mutant octamer oligonucleotide had little or no effect on cell death. These data are consistent with the hypothesis that cell death results from a cascade of cellular and molecular events and that an early event in programmed neuronal cell death is a decrease in binding of transcription factor(s) to octamer motif sequences.

Development of the mesencephalic nucleus of the trigeminal nerve in chick embryos: target innervation, neurotrophin receptors, and cell death.

The goal of this study was to determine whether processes of neurons in the mesencephalic nucleus of the trigeminal nerve (Mes V) of chick embryos arrive in their peripheral target prior to the period of developmental cell death, and to determine whether neurons with early target contact survive to a greater extent than neurons with processes that reach their peripheral target later. The arrival of Mes V nerve fibers in the masticatory muscles was determined by injecting the fluorescent tracer DiI, and the position of labeled and unlabeled neurons was mapped in subdivisions of the Mes V nucleus. Developmental changes in the numerical configuration of Mes V subdivisions were studied in DiI-labeled as well as Nissl-stained material. The expression of low-affinity (p75) neurotrophin receptors was investigated throughout development of the Mes V nucleus with in situ hybridization to assess whether and how levels of expression of this trophic receptor may relate to target innervation and cell death. The extent of cell death was evaluated by counting pyknotic nuclei. Processes of Mes V neurons invade their peripheral target between 5 and 7 days of incubation (E5-7). At E7-12, between 800 and 1,400 labeled Mes V neurons were distributed throughout the two main subdivisions of the Mes V nucleus, the tectal commissure and the optic tectum. Only few Mes V neurons were labeled in the posterior commissure or outside the brain. Cell counts in Nissl-stained material from E7-13 revealed that the numbers of Mes V neurons in the optic tectum decreased to about 40-60%, and in the tectal commissure to 20-25%, whereas Mes V neurons in the posterior commissure disappeared almost entirely. Few Mes V neurons remained in the leptomeninges at E8-10, but a considerable number was found outside the midbrain at E11, indicating ongoing migration of some Mes V neurons. Neurotrophin receptors were differentially expressed in the Mes V nucleus: Before and after the period of cell death, 90-100% of Mes V neurons expressed neurotrophin receptors, whereas during, and immediately preceding the period of developmental cell death (E9-E13), merely 70% of Mes V neurons expressed this receptor. These findings are consistent with the hypothesis that early target contact may provide an advantage for the survival of Mes V neurons and that competition for trophic factors may occur in the peripheral target of this nucleus prior to the period of cell death.

Lack of correspondence between mRNA expression for a putative cell death molecule (SGP‐2) and neuronal cell death in the central nervous system

Neuronal death during nervous system development, a widely observed phenomenon, occurs through unknown mechanisms. Recent evidence suggests an active, destructive process requiring new gene expression. Sulfated glycoprotein-2 (SGP-2), a secretory product of testicular Sertoli cells has been shown to up-regulate in several nonneural tissues undergoing programmed cell death and in several types of neuronal degeneration. In order to determine if this message up-regulates in neurons undergoing developmentally determined cell death, we have studied the expression of SGP-2 mRNA in the developing and adult rat central nervous system (CNS) with in situ hybridization. We also report on the expression of this message in nonneural tissues from several regions of the developing embryo. The developing and adult rat central nervous system as well as widely varied tissues in the rat embryo express SGP-2 mRNA in a pattern that does not correlate with regions undergoing developmental cell death. In the nervous system, SGP-2 mRNA is expressed in neuronal populations including motor neurons, cortical neurons, and hypothalamic neurons at ages when the period of developmental cell death has passed. In a nonneural tissue (palatal shelve epithelium) for which a developmental cell death period has been described, SGP-2 mRNA was not present in the region where cell death occurs. We conclude that SGP-2 mRNA expression cannot be correlated with programmed cell death in neural or nonneural tissues. The results of this study as well as recently reported SGP-2 homologies indicate a possible role for this protein in secretion and lipid transport.