Low density SCG cultures for neurite extension assays were coated with poly-d-lysine and laminin (both from Collaborative Biomedical Products, Bedford, MA). to activation time course and downstream targets, leading to selective regulation of neuritogenesis and survival. Such differential responsiveness to two ligands acting through the same Trk receptor has important implications for neurotrophin function throughout the nervous system. The neurotrophic factor hypothesis postulates that interactions between a Rabbit polyclonal to cox2 AL 8697 developing peripheral neuron and its target organ play an essential role in AL 8697 neuronal competition and cell death. This hypothesis is perhaps best exemplified by developing sympathetic neurons, which are absolutely dependent upon NGF, one member of the neurotrophin family of growth factors (7, 28, 42, 47), during the period of target competition (9, 43C45, 62). During this developmental window that occurs neonatally, NGF is believed to bind to its cognate receptors on the terminals of sympathetic neurons and to regulate their afferent input density via two primary mechanisms. First, NGF stimulates arborization and synaptogenesis via appropriate input neurons. Secondly, NGF serves as a discriminating mechanism that allows the elimination of neurons that have failed to sequester adequate target territory. This latter function is accomplished by an NGF-induced signal transduction cascade that prevents neuronal apoptosis (19, 20). Target-derived NGF initiates these responses by binding to two different cell surface receptors: the tyrosine kinase receptor TrkA (33, 34, 36), a member of the Trk family of receptors (2), and the p75 neurotrophin receptor (12). TrkA binds preferentially to NGF, but it can also bind the structurally related neurotrophin-3 (NT-3)1 (47) in 3T3 fibroblasts (15), while the p75 receptor binds all of the neurotrophins (56, 57). It is clear from studies on cultured neurons that NGF binding to TrkA alone is sufficient to mediate many of the prototypic biological responses (31). Moreover, all sympathetic neurons are lost in TrkA ?/? mice (61), as they are in NGF ?/? mice (16). However, the p75 receptor likely also plays a role since recent evidence indicates that it modulates TrkA tyrosine kinase activity (3, 71), that it signals on its own to modulate ceramide (22, 23) and NFB (10), and, finally, AL 8697 that there are deficits in sympathetic innervation in the p75 ?/? mice (41). Although the vast majority of sympathetic neurons AL 8697 have an absolute requirement for NGF during the period of target competition, three different lines of evidence indicate that these neurons may well see other cellular sources of neurotrophins, and that these other neurotrophins may play important biological roles. First, neurotrophins are made by Schwann cells (1, 30, 49), and sympathetic neurons themselves express both brain-derived neurotrophic factor (BDNF) and NT-3 mRNAs (60), raising the possibility of autocrine/paracrine interactions. Secondly, although neonatal sympathetic neurons do not respond to NT-3 with survival, as do their embryonic counterparts (8, 21), they express high affinity NT-3 binding sites (18); TrkC mRNA, which encodes the preferred Trk receptor for NT-3 (40), is definitely indicated at low levels in the neonatal superior cervical ganglion (SCG). Finally, NT-3 ?/? mice have 50% fewer sympathetic neurons (25, 26, 66) and display deficits in sympathetic target innervation that can be rescued by exogenous NT-3 (24). With this paper we demonstrate that NT-3 only very poorly supports the survival of NGF-dependent neonatal sympathetic neurons, but that when survival is managed by limiting quantities of NGF, NT-3 selectively mediates neuritogenesis and manifestation of genes associated with morphological growth. We have examined the biochemical basis of this differential biological responsiveness. Sympathetic neurons communicate relatively high levels of the TrkA receptor and low levels of the TrkC receptor. NGF activates TrkA inside a graded fashion, while NT-3 activates TrkA and, to a much lesser degree, TrkC. Both of these neurotrophins induce related sustained activation of TrkA, while NGF is definitely 10-fold more efficient than NT-3 in mediating short-term TrkA activity. This TrkA activation is necessary for NT-3 to mediate sympathetic neuron survival and neuritogenesis, as demonstrated using a mutant NT-3 that activates only TrkC. However, actually at related acute levels of TrkA activation, NT-3 mediates neuronal survival at levels two- to threefold less well than NGF. These data suggest that NGF and NT-3 differentially regulate the TrkA receptor both with regard to AL 8697 activation time program and downstream focuses on, leading to selective rules of neuritogenesis and survival. Such differential responsiveness to two ligands acting through the same Trk receptor offers important implications for neurotrophin function throughout the nervous system. Materials and Methods Main Neuronal.
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