Deciphering proneurotrophin actions.

Like most growth factors, neurotrophins are initially synthesized as precursors that are cleaved to release C-terminal mature forms. The well-characterized mature neurotrophins bind to Trk receptors to initiate survival and differentiative responses. More recently, the precursor forms or proneurotrophins have been found to act as distinct ligands by binding to an unrelated receptor complex consisting of the p75 neurotrophin receptor (p75) and sortilin to initiate cell death. Induction of proNGF and p75 has been observed in preclinical injury models and in pathological states in the central nervous system, and strategies that block the proNGF/p75 interaction are effective in limiting neuronal apoptosis. In contrast, the mechanisms that regulate expression of other proneurotrophins, including proBDNF and proNT-3, are less well understood. Here, recent findings on the biological actions, regulation of expression, and pathophysiological effects of proneurotrophins will be reviewed.

Brain-derived neurotrophic factor as a model system for examining gene by environment interactions across development.

There has been a dramatic rise in gene x environment studies of human behavior over the past decade that have moved the field beyond simple nature versus nurture debates. These studies offer promise in accounting for more variability in behavioral and biological phenotypes than studies that focus on genetic or experiential factors alone. They also provide clues into mechanisms of modifying genetic risk or resilience in neurodevelopmental disorders. Yet, it is rare that these studies consider how these interactions change over the course of development. In this paper, we describe research that focuses on the impact of a polymorphism in a brain-derived neurotrophic factor (BDNF) gene, known to be involved in learning and development. Specifically we present findings that assess the effects of genotypic and environmental loadings on neuroanatomic and behavioral phenotypes across development. The findings illustrate the use of a genetic mouse model that mimics the human polymorphism, to constrain the interpretation of gene-environment interactions across development in humans.

Secreted proNGF is a pathophysiological death-inducing ligand after adult CNS injury.

The unprocessed precursor of the neurotrophin nerve growth factor (NGF), proNGF, has been suggested to be a death-inducing ligand for the neurotrophin receptor p75. Whether proNGF is a true pathophysiological ligand that is secreted, binds p75, and activates cell death in vivo, however, has remained unknown. Here, we report that after brain injury, proNGF was induced and secreted in an active form capable of triggering apoptosis in culture. We further demonstrate that proNGF binds p75 in vivo and that disruption of this binding results in complete rescue of injured adult corticospinal neurons. These data together suggest that proNGF binding to p75 is responsible for the death of adult corticospinal neurons after lesion, and they help to establish proNGF as the pathophysiological ligand that activates the cell-death program by means of p75 after brain injury. Interference in the binding of proNGF to p75 may provide a therapeutic approach for the treatment of disorders involving neuronal loss.

Neurotrophins and their receptors: signaling trios in complex biological systems.

The neurotrophins, a class of four related growth factors, utilize a dual receptor system consisting of Trk receptor tyrosine kinases and the structurally unrelated p75(NTR) to modulate diverse and sometimes opposing biological actions. The identification of novel ligands for p75(NTR), unconventional mechanisms for Trk activation and unique signaling intermediates further underscores the complex nature of neurotrophin: receptor interactions, as well as their functions within and outside of the nervous systems. This review summarizes recent surprises of how ligand-receptor pairing may affect diverse developmental events, regulate response to injury and extend their influence on memory and learning.

Regulation of cell survival by secreted proneurotrophins.

Neurotrophins are growth factors that promote cell survival, differentiation, and cell death. They are synthesized as proforms that can be cleaved intracellularly to release mature, secreted ligands. Although proneurotrophins have been considered inactive precursors, we show here that the proforms of nerve growth factor (NGF) and the proforms of brain derived neurotrophic factor (BDNF) are secreted and cleaved extracellularly by the serine protease plasmin and by selective matrix metalloproteinases (MMPs). ProNGF is a high-affinity ligand for p75(NTR) with high affinity and induced p75NTR-dependent apoptosis in cultured neurons with minimal activation of TrkA-mediated differentiation or survival. The biological action of neurotrophins is thus regulated by proteolytic cleavage, with proforms preferentially activating p75NTR to mediate apoptosis and mature forms activating Trk receptors to promote survival.

The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor.

Ligand-induced receptor oligomerization is an established mechanism for receptor-tyrosine kinase activation. However, numerous receptor-tyrosine kinases are expressed in multicomponent complexes with other receptors that may signal independently or alter the binding characteristics of the receptor-tyrosine kinase. Nerve growth factor (NGF) interacts with two structurally unrelated receptors, the Trk A receptor-tyrosine kinase and p75, a tumor necrosis factor receptor family member. Each receptor binds independently to NGF with predominantly low affinity (K(d) = 10(-9) m), but they produce high affinity binding sites (K(d) = 10(-11) m) upon receptor co-expression. Here we provide evidence that the number of high affinity sites is regulated by the ratio of the two receptors and by specific domains of Trk A and p75. Co-expression of Trk A containing mutant transmembrane or cytoplasmic domains with p75 yielded reduced numbers of high affinity binding sites. Similarly, co-expression of mutant p75 containing altered transmembrane and cytoplasmic domains with Trk A also resulted in predominantly low affinity binding sites. Surprisingly, extracellular domain mutations of p75 that abolished NGF binding still generated high affinity binding with Trk A. These results indicate that the transmembrane and cytoplasmic domains of Trk A and p75 are responsible for high affinity site formation and suggest that p75 alters the conformation of Trk A to generate high affinity NGF binding.

Brain derived neurotrophic factor is an endothelial cell survival factor required for intramyocardial vessel stabilization.

Brain derived neurotrophic factor, BDNF, is a neurotrophin best characterized for its survival and differentiative effects on neurons expressing the trk B receptor tyrosine kinase. Although many of these neurons are lost in the BDNF(-)(/)(- )mouse, the early postnatal lethality of these animals suggests a wider function for this growth factor. Here, we demonstrate that deficient expression of BDNF impairs the survival of endothelial cells in intramyocardial arteries and capillaries in the early postnatal period, although the embryonic vasculature can remodel into arteries, capillaries and veins. BDNF deficiency results in a reduction in endothelial cell-cell contacts and in endothelial cell apoptosis, leading to intraventricular wall hemorrhage, depressed cardiac contractility and early postnatal death. Vascular hemorrhage is restricted to cardiac vessels, reflecting the localized expression of BDNF and trk B by capillaries and arterioles in this vascular bed. Conversely, ectopic BDNF overexpression in midgestational mouse hearts results in an increase in capillary density. Moreover, BDNF activation of endogenous trk B receptors supports the survival of cardiac microvascular endothelial cells cultured from neonatal mice. These results establish an essential role for BDNF in maintaining vessel stability in the heart through direct angiogenic actions on endothelial cells.

Trk C receptor signaling regulates cardiac myocyte proliferation during early heart development in vivo.

Neurotrophin-3 (NT-3) is a member of the neurotrophin family of growth factors, best characterized by its survival- and differentiation-inducing effects on developing neurons bearing the trk C receptor tyrosine kinase. Through analysis of NT-3 and trk C gene-targeted mice we have identified NT-3 as critically regulating cardiac septation, valvulogenesis, and conotruncal formation. Although these defects could reflect cardiac neural crest dysfunction, the expression of NT-3 and trk C by cardiac myocytes prior to neural crest migration prompted analysis of cell-autonomous actions of NT-3 on cardiac myocytes. Retroviral-mediated overexpression of truncated trk C receptor lacking kinase activity was used to inhibit activation of trk C by endogenous NT-3, during early heart development in ovo. During the first week of chicken development, expression of truncated trk C reduced myocyte clone size by more than 60% of control clones. Direct mitogenic actions of NT-3 on embryonic cardiac myocytes were demonstrated by analysis of BrdU incorporation or PCNA immunoreactivity in control and truncated trk C-expressing clones. Inhibition of trk C signaling reduced cardiac myocyte proliferation during the first week of development, but had no effect at later times. These studies demonstrate that endogenous NT-3:trk C signaling regulates cardiac myocyte proliferation during cardiac looping and the establishment of ventricular trabeculation but that myocyte proliferation becomes NT-3 independent during the second week of embryogenesis.

p75(NTR) mediates neurotrophin-induced apoptosis of vascular smooth muscle cells.

The development of atherosclerotic lesions results from aberrant cell migration, proliferation, and extracellular matrix production. In advanced lesions, however, cellular apoptosis, leading to lesion remodeling, predominates. During lesion formation, the neurotrophins and the neurotrophin receptor tyrosine kinases, trks B and C, are induced and mediate smooth muscle cell migration. Here we demonstrate that a second neurotrophin receptor, p75(NTR), is expressed by established human atherosclerotic lesions and late lesions that develop after balloon injury of the rat thoracic aorta. The p75(NTR), a member of the tumor necrosis factor/FAS receptor family, can modulate trk receptor function as well as initiate cell death when expressed in cells of the nervous system that lack kinase-active trk receptors. p75(NTR) expression colocalizes to neointimal cells, which express smooth muscle cell alpha-actin and are expressed by cultured human endarterectomy-derived cells (HEDC). Areas of the plaque expressing p75(NTR) demonstrate increased TUNEL positivity, and HEDC undergo apoptosis in response to the neurotrophins. Finally, neurotrophins also induced apoptosis of a smooth muscle cell line genetically manipulated to express p75(NTR), but lacking trk receptor expression. These studies identify the regulated expression of neurotrophins and p75(NTR) as an inducer of smooth muscle cell apoptosis in atherosclerotic lesions.

TrkA immunoglobulin-like ligand binding domains inhibit spontaneous activation of the receptor.

The extracellular region of the nerve growth factor (NGF) receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent receptor dimerization, chimeric receptors were generated by swapping the Ig-like domains of the TrkA receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit receptor dimerization in the absence of NGF.

Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis.

The Crk family of adaptor proteins participate in diverse signaling pathways that regulate growth factor-induced proliferation, anchorage-dependent DNA synthesis, and cytoskeletal reorganization, important for cell adhesion and motility. Using kidney epithelial 293T cells for transient co-transfection studies and the nerve growth factor (NGF)-responsive PC12 cell line as a model system for neuronal morphogenesis, we demonstrate that the non-receptor tyrosine kinase c-Abl is an intermediary for NGF-inducible c-Crk II phosphorylation on the negative regulatory Tyr(222). Transient expression of a c-Crk II Tyr(222) point mutant (c-Crk Y222F) in 293T cells induces hyperphosphorylation of paxillin on Tyr(31) and enhances complex formation between c-Crk Y222F and paxillin as well as c-Crk Y222F and c-Abl, suggesting that c-Crk II Tyr(222) phosphorylation induces both the dissociation of the Crk SH2 domain from paxillin and the Crk SH3 domain from c-Abl. Interestingly, examination of the early kinetics of NGF stimulation in PC12 cells showed that c-Crk II Tyr(222) phosphorylation preceded paxillin Tyr(31) phosphorylation, followed by a transient initial dissociation of the c-Crk II paxillin complex. PC12 cells overexpressing c-Crk Y222F manifested a defect in cellular adhesion and neuritogenesis that led to detachment of cells from the extracellular matrix, thus demonstrating the biological significance of c-Crk II tyrosine phosphorylation in NGF-dependent morphogenesis. Whereas previous studies have shown that Crk SH2 binding to paxillin is critical for cell adhesion and migration, our data show that the phosphorylation cycle of c-Crk II determines its dynamic interaction with paxillin, thereby regulating turnover of multiprotein complexes, a critical aspect of cytoskeletal plasticity and actin dynamics.

Trk C signaling is required for retinal progenitor cell proliferation.

Although neurotrophin actions in the survival of specific retinal cell types have been identified, the biological functions for neurotrophin-3 (NT-3) in early retinal development remain unclear. Having localized NT-3 and trk C expression at early developmental stages when retinal neuroepithelial progenitor cells predominate, we sought to modulate NT-3 signaling in these cells by overexpressing a truncated isoform of the NT-3 receptor, trk C. We have demonstrated that this non-catalytic receptor can inhibit NT-3 signaling when coexpressed with the full-length kinase-active trk C receptor. Using a replication-deficient retrovirus to ectopically express the truncated trk C receptor to limited numbers of progenitor cells in ovo, we examined the effects of disrupted trk C signaling on the proliferation or differentiation of retinal cells. Clones expressing truncated trk C exhibited a 70% reduction in clone size, compared with clones infected with a control virus, indicating that inhibition of trk C signaling decreased the clonal expansion of cells derived from a single retinal progenitor cell. Additionally, impaired NT-3 signaling resulted in a reduction of all retinal cell types, suggesting that NT-3 targets retinal precursor cells rather than differentiated cell types. BrdU labeling studies performed at E6 indicate that this reduction in cell number occurs through a decrease in cell proliferation. These studies suggest that NT-3 is an important mitogen early in retinal development and serves to establish the size of the progenitor pool from which all future differentiated cells arise.

Activation of c-Ha-Ras by nitric oxide modulates survival responsiveness in neuronal PC12 cells.

p21(c-Ha-Ras) (Ras) can be activated by the guanine nucleotide exchange factor mSOS1 or by S-nitrosylation of cysteine 118 via nitric oxide (NO). To determine whether these two Ras-activating mechanisms modulate distinct biological effects, a NO-nonresponsive Ras mutant (Ras(C118S)) was stably expressed in the PC12 cells, a cell line that generates NO upon nerve growth factor treatment. We report here that Ras(C118S) functions indistinguishably from wild type Ras in activating and maintaining the mSOS1- and Raf-1-dependent mitogen-activated protein kinase cascade necessary for neuronal differentiation. However, continuous (>5 days) exposure to nerve growth factor reveals that, in contrast to parental or wild-type Ras-overexpressing PC12 cells, Ras(C118S)-expressing PC12 cells cannot sustain the basal interaction between Ras and phosphatidylinositol 3-kinase. This results in spontaneous apoptosis of these cells despite the presence of nerve growth factor and serum. Thus unique downstream effector interactions and biological outcomes can be differentially modulated by distinct modes of Ras activation.

Regulation of cardiac development by receptor tyrosine kinases.

The development of a functional heart depends on the coordinated growth, differentiation, migration, and apoptosis of cell populations of diverse embryological origins. These processes are regulated in part by soluble polypeptide growth factors that exert their effects via binding to cell surface receptors with intrinsic tyrosine kinase activity. In particular, members of this class of receptors and their ligands have been shown to regulate the development of distinctive regions of the heart, such as the mesodermally derived cardiac myocyte, the endocardium, and outflow tract and septa, which depend on cardiac neural crest. The hepatocyte growth factor receptor, c-met the fibroblast growth factor receptors; and the neuregulin receptors have been shown to influence cardiomyocyte proliferation and/or differentiation. Receptors binding to vascular endothelial cell growth factor or angiopoietin have been implicated in the development of the endocardium. Finally, gene-targeting experiments in the mouse have demonstrated functional roles for neurotrophins and their cognate trk receptor tyrosine kinases in the development of outflow tract, septa, and valves that are structures derived from cardiac neural crest.

Immunohistochemical analysis of the neurotrophins BDNF and NT-3 and their receptors trk B, trk C, and p75 in the developing chick retina.

The neurotrophins are trophic and mitogenic factors critical for the development of specific classes of neurons in the central and peripheral nervous systems. In the retina, BDNF and NT-3 have been shown to promote the survival of differentiated ganglion cells (Rodriguez-Tebar et al., 1989; De La Rosa et al., 1994). NT-3 has also been demonstrated to support the survival of amacrine cells and facilitates the differentiation of retinal neurons in culture (De La Rosa et al., 1994). Here, we examine immunohistochemically the expression of BDNF and NT-3 proteins, their cognate receptors, trk B and trk C, respectively, and the p75 neurotrophin receptor in the developing chick retina. At E8, the earliest stage of retinal development examined, all of these proteins exhibit diffuse expression throughout the width of the retina, with the strongest reactivity in the innermost layers. A gradual restriction in expression to ganglion cells and amacrine cells, the staining of which is most prominent at E15, is followed by a downregulation of expression with the strongest immunoreactivity persisting in the ganglion cell layer. Overlapping patterns of expression throughout embryonic development indicate a colocalization of the neurotrophins and their receptors, although NT-3 and p75 alone are present in the inner plexiform layer and only p75 is observed in the outer plexiform layer. Although some of the immunoreactivity for BDNF, NT-3, and their receptors in retina may reflect trophic mechanisms operating in association with the optic tectum and isthmo-optic nucleus, the colocalization of ligands and receptors in retina strengthens the assertion that these neurotrophins function locally during development.

A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction.

We have identified the site of molecular interaction between nitric oxide (NO) and p21(ras) responsible for initiation of signal transduction. We found that p21(ras) was singly S-nitrosylated and localized this modification to a fragment of p21(ras) containing Cys118. A mutant form of p21(ras), in which Cys118 was changed to a serine residue and termed p21(ras)C118S, was not S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21(ras), resulting in an active form, but not on p21(ras)C118S. Furthermore, in contrast to parental Jurkat T cells, NO-related species did not stimulate mitogen-activated protein kinase activity in cells transfected with p21(ras)C118S. These data indicate that Cys118 is a critical site of redox regulation of p21(ras), and S-nitrosylation of this residue triggers guanine nucleotide exchange and downstream signaling.

v-Crk, an effector of the nerve growth factor signaling pathway, delays apoptotic cell death in neurotrophin-deprived PC12 cells.

v-Crk is a member of a class of SH2 and SH3-containing adaptor proteins that have been implicated in regulating the TrkA receptor tyrosine kinase and potentiating Nerve Growth Factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells (Hempstead et al, Mol. Cell Biol. 14: 1964 - 1971). Given the fact that NGF induces both differentiation and survival by binding to TrkA, we examined the rate of apoptotic cell death elicited by NGF-withdrawal in native, v-Crk, and TrkA-expressing PC12 cells. While more than 50% of native PC12 cells underwent apoptosis within 48 h of NGF withdrawal, the v-Crk and TrkA-expressing cells were much more resistant to apoptosis under these conditions, whereby approximately 70 and 95%, respectively, of the cells were alive. The ability of v-Crk to delay apoptosis required prior NGF-dependent differentiation, since naive undifferentiated v-Crk expressing PC12 cells or cells that express v-Crk mutants that are defective in NGF signaling were not protected from apoptosis during growth factor withdrawal. Moreover, addition of 50 ng/ml EGF to serum and NGF deprived v-Crk expressing cells, which also causes neurite outgrowth, promoted complete and long-term survival, although such EGF replacement had no neurotrophic effect on wild-type PC12 cells or PC12 cells overexpressing Human Bcl-2. These experiments suggest that v-Crk potentiation of a receptor tyrosine kinase under conditions of growth factor deprivation is essential for preventing apoptosis. However, unlike native PC12 cells, neither v-Crk or TrkA-expressing PC12 cells exhibited a G1 arrest when incubated for 2 weeks in NGF. Thus, v-Crk and TrkA may protect NGF deprived PC12 by preventing cell cycle arrest and hence an aborted entry into a defective cell cycle. Moreover, during NGF-withdrawal, v-CrkPC12 cells exhibited down regulation in MAP kinase and JNK activities while in native cells, these activities increased within 6 - 8 h after NGF deprivation. Thus, unlike v-Crk-mediated augmentation of differentiation, sustained activation of MAP kinase may not be required for v-Crk-induced cell survival.

Targeted deletion of all isoforms of the trkC gene suggests the use of alternate receptors by its ligand neurotrophin-3 in neuronal development and implicates trkC in normal cardiogenesis.

We have generated null mutant mice that lack expression of all isoforms encoded by the trkC locus. These mice display a behavioral phenotype characterized by a loss of proprioceptive neurons. Neuronal counts of sensory ganglia in the trkC mutant mice reveal less severe losses than those in NT-3 null mutant mice, strongly suggesting that NT-3, in vivo, may signal through receptors other than trkC. Mice lacking either NT-3 or all trkC receptor isoforms die in the early postnatal period. Histological examination of trkC-deficient mice reveals severe cardiac defects such as atrial and ventricular septal defects, and valvular defects including pulmonic stenosis. Formation of these structures during development is dependent on cardiac neural crest function. The similarities in cardiac defects observed in the trkC and NT-3 null mutant mice indicate that the trkC receptor mediates most NT-3 effects on the cardiac neural crest.

Identification of an essential nonneuronal function of neurotrophin 3 in mammalian cardiac development.

Neurotrophin 3 (Nt3) is one of five neurotrophin growth factors which shape the development of the nervous system by regulating neuronal survival and differentiation. Peripheral neuronal subpopulations expressing the TrkC receptor tyrosine kinase respond to Nt3 with enhanced survival, mitogenesis or cell migration and these neurons are lost in homozygous Nt3 null (-/-) mutant mice. The unexplained perinatal lethality in the Nt3-/- mice, however, suggests a wider function for this neurotrophin. Here we report that Nt3 is essential for the normal development of atria, ventricles, and cardiac outflow tracts. Histological and echocardiographic image analysis of Nt3-/- animals reveal severe cardiovascular abnormalities including atrial and ventricular septal defects, and tetralogy of Fallot, resembling some of the most common congenital malformations in humans. The observed defects are consistent with abnormalities in the survival and/or migration of cardiac neural crest early in embryogenesis and establish an essential role for neurotrophin 3 in regulating the development of the mammalian heart.