Developmentally regulated expression of persyn, a member of the synuclein family, in skin.

Synucleins constitute a group of unique, evolutionarily conserved proteins that are expressed predominantly in neurons of the central and peripheral nervous system. Although the normal cellular functions of synucleins are not clear, these proteins have been implicated in various neurodegenerative conditions in humans. We found that persyn, a recently characterized member of the synuclein family, is expressed not only in the nervous system but also in the stratum granulosum of the epidermis of neonatal and adult mice. This finding together with our recent observations that persyn influences neurofilament network integrity in sensory neurons raises the possibility that persyn in skin could be involved in modulation of the keratin network.

Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system.

The synucleins are a unique family of small intracellular proteins that have recently attracted considerable attention because of their involvement in human neurodegenerative diseases. We have cloned a new member of the synuclein family called persyn. In contrast to other synucleins, which are presynaptic proteins of CNS neurons, persyn is a cytosolic protein that is expressed predominantly in the cell bodies and axons of primary sensory neurons, sympathetic neurons, and motoneurons. Northern blotting, in situ hybridization, Western blotting, and immunohistochemistry revealed that persyn mRNA and protein are expressed in these neurons from the earliest stages of axonal outgrowth and are maintained at a high level throughout life. Persyn also becomes detectable in evolutionary recent regions of the brain by adulthood.

GFRalpha-4, a new GDNF family receptor.

GFRalpha-1, GFRalpha-2, and GFRalpha-3 constitute a family of structurally related, glycosyl-phosphatidylinosital-linked, cell surface proteins, two of which, GFRalpha-1 and GFRalpha-2, are components of the receptor complex for the neurotrophic factors GDNF and neurturin, respectively. By screening an embryonic chicken brain cDNA library with a GFRalpha-1 probe at low stringency, we isolated cDNAs encoding an additional member of the GFRalpha family, GFRalpha-4. The nucleotide sequence predicts a 431-amino-acid secreted protein that is more closely related to GFRalpha-1 and GFRalpha-2 than to GFRalpha-3. GFRalpha-4 mRNA is expressed in distinctive patterns in the brain and several other organs and tissues of the chicken embryo. Our findings extend the family of GFRalpha proteins and provide information about the tissues in which GFRalpha-4 may function during development.

Bcl-2 accelerates the maturation of early sensory neurons.

Bcl-2 is a cytoplasmic protein that blocks apoptosis in a wide variety of cell types. Here we report a novel role for Bcl-2 in the early stages of neuronal development. Shortly after differentiating from progenitor cells, sensory neurons undergo a distinct morphological change; initially they have small, spindle-shaped, phase-dark cell bodies that become large, spherical, and phase-bright. Early sensory neurons cultured from the trigeminal ganglia of bcl-2-/- embryos at embryonic day 11 (E11) and E12 underwent this change more slowly than trigeminal neurons of wild-type embryos of the same ages. The delay was not attributable to the well documented role of Bcl-2 in preventing apoptosis, because Bcl-2-deficient early sensory neurons survived as well as wild-type neurons. Accordingly, there was a significantly smaller number of the more mature type of neuron in the early trigeminal ganglia of bcl-2-/- embryos, yet the number of neurons in the trigeminal ganglia of bcl-2-/- and wild-type embryos was similar. The absence of Bcl-2 did not cause a uniform delay in the developmental program of sensory neurons, because the time course of nerve growth factor receptor expression (both trkA and p75) was unaffected in the trigeminal neurons of bcl-2-/- embryos. These findings indicate that Bcl-2 expression is required for the normal progression of a particular early maturational change in embryonic sensory neurons.

Bcl-2 is required for cranial sensory neuron survival at defined stages of embryonic development.

To ascertain the role of endogenous Bcl-2 in maintaining the survival of developing neurons and modulating their responses to neurotrophins, we compared the in vitro and in vivo survival of cranial sensory neurons of wild-type and bcl-2 null mouse embryos. At the peak of naturally occurring neuronal death in the trigeminal ganglion at E14, trigeminal neurons from bcl-2(-/-) embryos initially survived in culture in response to NGF but were not sustained as well as neurons from wild-type embryos. At the end of the period of naturally occurring neuronal death at E18, Bcl-2-deficient trigeminal neurons survived with NGF as well as wild-type neurons. At E14 in vivo, the number of trigeminal neurons undergoing apoptosis was significantly greater in bcl-2(-/-) embryos, and there were significantly fewer neurons in the trigeminal ganglia of bcl-2(-/-) embryos at E16 and E18. Similar age-related changes in the responses of nodose ganglion neurons to BDNF were observed in cultures established from bcl-2(-/-) and wild-type embryos between E14 and E18. These results suggest that endogenous Bcl-2 is required for the sustained survival response of a subset of cranial sensory neurons to neurotrophins at particular stages of embryonic development and show that its absence leads to reduced numbers of these neurons in vivo.

Neurturin responsiveness requires a GPI-linked receptor and the Ret receptor tyrosine kinase.

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor (GDNF). Both factors promote the survival of a variety of neurons, and GDNF is required for the development of the enteric nervous system and kidney. GDNF signals through a receptor complex consisting of the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)-linked receptor termed GDNFR-alpha. Here we report the cloning of a new GPI-linked receptor termed NTNR-alpha that is homologous with GDNFR-alpha and is widely expressed in the nervous system and other tissues. By using microinjection to introduce expression plasmids into neurons, we show that coexpression of NTNR-alpha with Ret confers a survival response to neurturin but not GDNF, and that coexpression of GDNFR-alpha with Ret confers a survival response to GDNF but not neurturin. Our findings indicate that GDNF and neurturin promote neuronal survival by signalling through similar multicomponent receptors that consist of a common receptor tyrosine kinase and a member of a GPI-linked family of receptors that determines ligand specificity.

Sympathetic neuron survival and TrkA expression in NT3-deficient mouse embryos.

Several in vitro and in vivo studies have led to the widely accepted view that NT3 is required for sympathetic neuroblast survival, induction of TrkA expression and the acquisition of NGF dependence. However, we show that the number of neurons and the levels of trkA and p75 mRNAs in the superior cervical sympathetic ganglion (SCG) of NT3-/- mouse embryos increase normally up to E16, 2 days after SCG neurons start responding to NGF. At E18 and in the postnatal period, there are significant reductions in the number of SCG neurons and in the levels of trkA and p75 mRNAs. These results show that the neurotrophin survival requirements of SCG neurons do not switch from NT3 to NGF during development and that NT3 is not required for the expression of TrkA and p75 and the acquisition of NGF dependence. Rather, some sympathetic neurons have a late requirement for NT3 at the time when they also depend on NGF for survival. The expression of transcripts encoding catalytic TrkC is negligible at this stage, suggesting that NT3 acts mainly via TrkA.

Expression and function of TrkB variants in developing sensory neurons.

Mouse trigeminal neurons survive independently of neurotrophins when their axons are growing to their targets, and are then transiently supported by BDNF before becoming NGF dependent. During the stage of neurotrophin independence, transcripts encoding the BDNF receptor, TrkB, were expressed at very low levels. During the stage of BDNF dependence, high levels of a transcript encoding a receptor with the catalytic tyrosine kinase domain were expressed. Although the levels of this transcript fell as the neurons lost responsiveness to BDNF, there were concomitant increases in the expression of transcripts encoding TrkB variants lacking the kinase domain. Analysis of RNA from purified neurons showed that all of these transcripts were present in neurons. BDNF and NGF up-regulated the expression of these transcripts early in development but had little effect later on. To test whether truncated TrkB modulates BDNF signalling via catalytic TrkB, we injected TrkB expression plasmids into NGF-dependent sympathetic neurons. Whereas expression of catalytic TrkB alone conferred a BDNF survival response, co-expression of non-catalytic TrkB substantially reduced this response. Our results suggest that BDNF responsiveness in sensory neurons during development is modulated by the relative levels of catalytic and non-catalytic TrkB.

Timing of neuronal death in trkA, trkB and trkC mutant embryos reveals developmental changes in sensory neuron dependence on Trk signalling.

The sensory neurons of the embryonic mouse trigeminal ganglion are supported in culture by different neurotrophins at successive stages of development. Initially the neurons survive in response to BDNF and NT3 and later switch to becoming NGF-dependent (Buchman, V. I. and Davies, A. M. (1993), Development 118, 989-1001). To determine if this in vitro switch in neurotrophin responsiveness is physiologically relevant, we studied the timing of neuronal death in the trigeminal ganglia of embryos that are homozygous for null mutations in the trkA, trkB and trkC genes, which encode receptor tyrosine kinases for NGF, BDNF and NT3, respectively. In wild-type embryos, the number of pyknotic nuclei increased from E11 to peak between E13 and E14, and decreased gradually at later ages, becoming negligible by birth. Neuronal death in the trigeminal ganglia of trkA-/- embryos also peaked between E13 and E14, but was almost threefold greater than in wild-type embryos at this stage. Whereas there was no significant difference between the number of pyknotic nuclei in trkA-/- and wild-type embryos at E11 and E12, there was a substantial increase in the number of pyknotic nuclei in the trigeminal ganglia of trkB-/- at these earlier stages. Counts of the total number of neurons in E13 trigeminal ganglia revealed a marked decrease in trkB-/- but not trkA-/- or trkC-/- embryos. Consistent with the later onset of excessive neuronal death in trkA-/- embryos, there was a marked decrease in the neuronal complement of the trigeminal ganglia of trkA-/- embryos at E15. These results demonstrate that TrkB signalling is required for the in vivo survival of many trigeminal neurons during the early stages of target field innervation before they become NGF-dependent.

Target and afferents interact to control developmental cell death in the mesencephalic parabigeminal nucleus of the rat.

During the period of natural cell death in the developing mammalian brain, both target cells and afferents have been shown to be important for neuronal survival. Here we demonstrate that afferents and targets have interactive roles in the maintenance of cells during development of the mesencephalic parabigeminal nucleus (PB) in rats. Pyknotic nuclei were counted in the PB of developing rats that received a bilateral lesion of the superior colliculus on the day of birth (P0). We observed that simultaneous deafferentation and deeferentation leads to a large peak of cell death at P1-2 in all three divisions of PB. Later the rate of pyknosis decreases and a second period of elevated cell death is observed just before the complete disappearance of the nucleus at P7-8. Counts of healthy neurones indicates two separate periods of increased neuronal loss. The first period occurs at P1-2, and the last and dramatic episode of cell loss at P8 leads to the disappearance of the PB. The combined effects of simultaneous target removal and deafferentation were different from the sum of the individual effects, indicating that the axonal targets and the afferents interact to control cell survival in the PB.

Intracellular compartmentalization of two differentially spliced s-rex/NSP mRNAs in neurons.

Using a subtractive hybridization technique directed to cloning transcripts with compartmentalized distributions within cerebral cortex neurons, we have isolated rat s-rex mRNAs that are analogues of the human neuroendocrine-specific NSP gene transcripts. Differential splicing produces two main s-rex mRNA that have different regional distributions in the developing and mature rat nervous system. In certain populations of adult brain neurons, most of s-rexs, mRNA and a substantial amount of s-rexb mRNA are localized to the axonal pole of the cell body. The localization of S-Rex/NSP proteins in these neurons suggests that s-rex mRNA compartmentalization targets the encoded proteins to specific regions of the neuron.

High specificity of neurotrophins in the embryonic chicken trigeminal system.

Studies of cell lines and some cultured neurons have demonstrated potential cross-talk between neurotrophins and their receptors; high concentrations of neurotrophins can exhibit either agonist or antagonistic actions on heterologous neurotrophin receptors. We have studied neurotrophin discrimination among the sensory neurons of the embryonic chicken trigeminal system. We show that nerve growth factor (NGF) at a concentration that is six orders of magnitude greater than that required to promote the survival of NGF-dependent dorsomedial trigeminal ganglion (DMTG) neurons has no effect on the survival of brain-derived neurotrophic factor (BDNF)-dependent trigeminal mesencephalic nucleus (TMN) neurons and does not affect the dose-response relationship of these neurons to BDNF. A similar high level of neurotrophin-3 neither promotes the survival of BDNF-dependent ventrolateral trigeminal ganglion neurons nor affects the dose response of these neurons to BDNF. High levels of BDNF have a negligible effect on the survival of mid-embryonic DMTG neurons. These results show that some neurons are able to discriminate completely between neurotrophins at very high concentrations, indicating that neurotrophin responses can be far more highly specific than previously appreciated.

The survival of NGF-dependent but not BDNF-dependent cranial sensory neurons is promoted by several different neurotrophins early in their development.

Recent work has shown that the survival of the nerve growth factor (NGF)-dependent trigeminal ganglion neurons of the mouse embryo is promoted by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) during the early stages of target field innervation (Buchman and Davies, (1993) Development, 118, 989-1001). The present study was undertaken to ascertain if responsiveness to multiple neurotrophins is a universal feature of the early stages of neuronal development or is restricted to only certain kinds of neurons. To address this issue, we took advantage of the accessibility, from an early developmental stage, of several populations of cranial sensory neurons in the chicken embryo that depend for survival on just one or two known neurotrophins during the phase of naturally occurring cell death. During the mid-embryonic period (E10 to E12) when the number of sensory neurons is declining due to naturally occurring neuronal death, the neurons of the jugular ganglion and the dorsomedial part of the trigeminal ganglion (DMTG) were supported by NGF, the neurons of the ventrolateral part of the trigeminal ganglion (VLTG) were supported by BDNF and the nodose ganglion contained a major subset of neurons supported by BDNF and a minor subset supported by NT-3. Earlier in development (E6), the survival of DMTG and jugular neurons was additionally promoted by BDNF and NT-3. In contrast, E6 VLTG neurons did not exhibit a survival response to either NGF or NT-3, and E6 nodose neurons did not exhibit a survival response to NGF.(ABSTRACT TRUNCATED AT 250 WORDS)

The pearl mutation accelerates the schedule of natural cell death in the early postnatal retina.

The time of maximal occurrence of pyknotic nuclei in the retinal ganglion cell layer of postnatal pearl mutant mice is earlier than that in normal mice (Linden and Pinto 1985). Both ganglion and displaced amacrine cells and glia populate the ganglion cell layer. Thus, in order to show that ganglion cells themselves are affected, we counted the numbers of surviving axons in the optic nerve of postnatal day (PND) 0, 4, 12 and adult mice. On PND 0, pearl mutant mice had 139,000 +/- 2800 (SEM) optic axons, about 8% more than wild-type mice (128,000 +/- 1,700; p = 0.031) but on PND 4, pearl mutants had 24% fewer axons than wild-type mice (96,000 +/- 3700 and 119,000 +/- 4600, respectively; p = 0.008). Thus, pearl mutants lose nearly five times as many retinal ganglion cells as wild-type mice in the interval from PND 0 to 4. The number of axons present in adult mice was nearly equal (56,700 +/- 3200 for wild-type and 52,500 +/- 2700 for pearl mutants p = 0.37). We searched for evidence for changes in the schedule of cell death among other neurons of the retina by counting the number of pyknotic nuclei in the various retinal layers. On PND 4, pearl mutant mice had more pyknotic nuclei in the neuroblastic layer than wild-type mice (5000 +/- 400 and 3900 +/- 300, respectively; p less than 0.05). The time-course of the appearance of pyknotic nuclei in the outer nuclear layer differed for the two genotypes (ANOVA, F = 12.5, p less than 0.001). The most striking difference was a greater number of pyknotic nuclei on PND 20 for the pearl mutants (1300) than for wild-type (480; p = 0.002). However, the total number of photoreceptors in adults did not differ between the two genotypes (3.6 x 10(6) +/- 2.4 x 10(5) for wild-type and 3.7 x 10(6) +/- 3.3 x 10(5) for pearl; p greater than 0.8). These results, taken together, show that natural cell death occurs at an earlier time for retinal ganglion cells of pearl mutants, but that the total number of retinal neurons surviving to adulthood is not affected appreciably by the mutation.

Interactive effects of deafferentation and target removal on cell death in the parabigeminal nucleus of developing rats.

This study was designed to test the effects of simultaneous deafferentation and target removal on cell death in the parabigeminal nucleus. Bilateral lesions of the superior colliculus were made in newborn rats and neuron death was evaluated in the dorsal (PBd), middle (PBm) and ventral (PBv) divisions of the nucleus. When the results of the bilateral lesions were compared with the effects of unilateral lesions reported in a previous study simultaneous deafferentation and target removal were found to produce an increase in the rate of cell death greater than, and with a time course differing from that, predicted by the sum of the separate effects of removal of afferents or targets. These data suggest that the trophic effects of afferents and targets interact during the period of naturally occurring cell death.

Interactive effects of deafferentation and target removal on cell death in the parabigeminal nucleus of developing rats

This study was designed to test the effects of simultaneous deafferentation and target removal on cell death in the parabigeminal nucleus. Bilateral lesions of the superior colliculus were made in newborn rats and neuron death was evaluated in the dorsal (PBd), middle (PBm) and ventral PBv) division of the nucleous. When the results of the bilateral lesions were compared with the effects of unilateral lesions reported in a previous study simultaneous deafferentation and tarfet removal were found to produce an increase in the rate of cell death greater than, and with a time course differing from that, predicted by the sun of the separate effects of removal of afferents or targets. These data suggest that the trophic effects of afferents and targets interact during the period of naturally occurring cell death

Dual control by targets and afferents of developmental neuronal death in the mammalian central nervous system: a study in the parabigeminal nucleus of the rat.

Natural and induced cell degeneration were studied in the mesencephalic parabigeminal nucleus of postnatally developing rats. Natural cell death in the normal parabigeminal nucleus had already started at birth, was maximal at 3 days, and proceeded with a declining rate until postnatal days 8-10 in the dorsal, middle, and ventral divisions that compose the nucleus. The number of neurons declined by approximately one-third between birth and postnatal day 15. A unilateral lesion of the superior colliculus made at birth modified this pattern. In the deafferented ipsilateral middle division, the rate of cell death was above normal from day 1 to day 10, and the number of neurons at day 15 was 60% less than in unoperated controls. In the contralateral middle division, in which at least some of the neurons were axotomized by the lesion, the rate of cell death increased at days 1-2 and decreased below normal at days 3-5. Induced changes in the number of neurons were consistent with this pattern, and at day 15 the number was similar to the control value. In the ipsilateral dorsal and ventral divisions, which suffered simultaneous axotomy and deafferentation, the rate of cell death increased in 2 peaks at days 1-2 and 4-6, and the numbers of neurons dropped to negligible values at day 15. The frequency curves of degenerating cells were poor predictors of the absolute changes in neuron numbers, and evidence was found of continued postnatal migration of neurons into the developing parabigeminal nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)