SH2-B and APS are multimeric adapters that augment TrkA signaling.

Neurotrophins influence growth and survival of sympathetic and sensory neurons through activation of their receptors, Trk receptor tyrosine kinases. Previously, we identified Src homology 2-B (SH2-B) and APS, which are structurally similar adapter proteins, as substrates of Trk kinases. In the present study, we demonstrate that both SH2-B and APS exist in cells as homopentamers and/or heteropentamers, independent of Trk receptor activation. Structure-function analyses revealed that the SH2-B multimerization domain resides within its amino terminus, which is necessary for SH2-B-mediated nerve growth factor (NGF) signaling. Overexpression of SH2-B enhances both the magnitude and duration of TrkA autophosphorylation following exposure of PC12 cells to NGF, and this effect requires the amino-terminal multimerization motif. Moreover, the amino terminus of SH2-B is necessary for TrkA/SH2-B-mediated morphological differentiation of PC12 cells. Together, these results indicate that the multimeric adapters SH2-B and APS influence neurotrophin signaling through direct modulation of Trk receptor autophosphorylation.

Differential neuronal localizations and dynamics of phosphorylated and unphosphorylated type 1 inositol 1,4,5-trisphosphate receptors.

Type 1 inositol 1,4,5-trisphosphate receptors are phosphorylated by cyclic-AMP-dependent protein kinase A at serines 1589 and 1755, with serine 1755 phosphorylation greatly predominating in the brain. Inositol 1,4,5-trisphosphate receptor protein kinase A phosphorylation augments Ca(2+) release. To assess type 1 protein kinase A phosphorylation dynamics in the intact organism, we developed antibodies selective for either serine 1755 phosphorylated or unphosphorylated species. Immunohistochemical studies reveal marked variation in localization. For example, in the hippocampus the phosphorylated type 1 inositol 1,4,5-trisphosphate receptor is restricted to CA1, while the unphosphorylated receptor occurs ubiquitously in CA1-CA3 and dentate gyrus granule cells. Throughout the brain the phosphorylated type 1 inositol 1,4,5-trisphosphate receptor is selectively enriched in dendrites, while the unphosphorylated receptor predominates in cell bodies. Focal cerebral ischemia in rats and humans is associated with dephosphorylation of type 1 inositol 1,4,5-trisphosphate receptors, and glutamatergic excitation of cerebellar Purkinje cells mediated by ibogaine elicits dephosphorylation of type 1 inositol 1,4,5-trisphosphate receptors that precedes evidence of excitotoxic neuronal degeneration. We have demonstrated striking variations in regional and subcellular distribution of inositol 1,4,5-trisphosphate receptor phosphorylation that may influence normal physiological intracellular Ca(2+) signaling in rat and human brain. We have further shown that the subcellular distribution of inositol 1,4,5-trisphosphate receptor phosphorylation in neurons is regulated by excitatory neurotransmission, as well as excitotoxic insult and neuronal ischemia-reperfusion. Phosphorylation dynamics of type 1 inositol 1,4,5-trisphosphate receptors may modulate intracellular Ca(2+) release and influence the cellular response to neurotoxic insults.

Spatially and functionally distinct roles of the PI3-K effector pathway during NGF signaling in sympathetic neurons.

NGF is a target-derived growth factor for developing sympathetic neurons. Here, we show that application of NGF exclusively to distal axons of sympathetic neurons leads to an increase in PI3-K signaling in both distal axons and cell bodies. In addition, there is a more critical dependence on PI3-K for survival of neurons supported by NGF acting exclusively on distal axons as compared to neurons supported by NGF acting directly on cell bodies. Interestingly, PI3-K signaling within both cell bodies and distal axons contributes to survival of neurons. The requirement for PI3-K signaling in distal axons for survival may be explained by the finding that inhibition of PI3-K in the distal axons attenuates retrograde signaling. Therefore, a single TrkA effector, PI3-K, has multiple roles within spatially distinct cellular locales during retrograde NGF signaling.

Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism.

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca(2+) entry through voltage-sensitive Ca(2+) channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca(2+). Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca(2+) response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca(2+)-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.

Spatial considerations for stimulus-dependent transcription in neurons.

Most neurons have elaborate dendrites as well as an axon emanating from the cell body that form synaptic connections with one or many target cells, which may be located a considerable distance from the cell body. Such complex and impressive morphologies allow some types of neurons to integrate inputs from one to many thousands of pre-synaptic partners and to rapidly propagate electrical signals, often over long distances, to post-synaptic target cells. Much slower, non-electrical signals also propagate from dendrites and distal axons to neuronal nuclei that influence survival, growth, and plasticity. The distances between distal dendrites and/or distal axons and cell bodies of neurons can be hundreds of microns to more than one meter. This long-range biochemical signal propagation from distal dendrites and distal axons to neuronal nuclei is entirely unique to neurons. This review is focused on excitatory neurotransmitter signaling from dendritic synapses to neuronal nuclei as well as on retrograde growth factor signaling from distal axons to neuronal nuclei.

Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.

Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. We have generated mice with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing us to assess the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. Our results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections.

Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons.

Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. CREB-mediated gene expression was both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 was activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. Overexpression of Bcl-2 reduced the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor-dependent expression of genes encoding prosurvival factors.

Characterization of an NGF-P-TrkA retrograde-signaling complex and age-dependent regulation of TrkA phosphorylation in sympathetic neurons.

Nerve growth factor (NGF) is a target-derived trophic factor for developing sympathetic and cutaneous sensory neurons. NGF promotes growth and survival of neurons via activation of the receptor tyrosine kinase TrkA. We used compartmentalized cultures of sympathetic neurons to address the mechanism of NGF signaling from distal axons and terminals to proximal axons and cell bodies. Our results demonstrate that an NGF-phospho-TrkA (NGF-P-TrkA)-signaling complex forms in distal axons and is retrogradely transported as a complex to cell bodies of sympathetic neurons. Although a minor fraction of both NGF and TrkA is retrogradely transported, a large fraction of the NGF that is retrogradely transported is found complexed with retrogradely transported TrkA. Interestingly, the metabolism of the P-TrkA complex is dramatically different in young, NGF-dependent sympathetic neurons as compared to older, NGF-independent sympathetic neurons. After withdrawal of NGF from distal axons of young neurons, P-TrkA within distal axons, as well as within proximal axons and cell bodies, dephosphorylates rapidly. In contrast, after withdrawal of NGF from distal axons of older neurons, P-TrkA within distal axons dephosphorylates completely, although more slowly than that in young neurons, whereas dephosphorylation of P-TrkA within proximal axons and cell bodies occurs markedly more slowly, with at least one-half of the level of P-TrkA remaining 2 d after NGF withdrawal. Thus, P-TrkA within the cell bodies of young, NGF-dependent sympathetic neurons is derived from distal axons. A more stable P-TrkA complex within cell bodies of mature sympathetic neurons may contribute to the acquisition of NGF independence for survival of mature sympathetic neurons.

A late phase of cerebellar long-term depression requires activation of CaMKIV and CREB.

Recently, it has been shown that cerebellar LTD has a late phase that may be blocked by protein synthesis inhibitors. To understand the mechanisms underlying the late phase, we interfered with the activation of transcription factors that might couple synaptic activation to protein synthesis. Particle-mediated transfection of cultured Purkinje neurons with an expression vector encoding a dominant inhibitory form of CREB resulted in a nearly complete blockade of the late phase. Kinases that activate CREB were inhibited, and LTD was assessed. Inhibition of PKA or the MAPK/RSK cascades were without effect on the late phase, while constructs designed to interfere with CaMKIV function attenuated the late phase. These results indicate that the activation of CaMKIV and CREB are necessary to establish a late phase of cerebellar LTD.

Patterning of cortical efferent projections by semaphorin-neuropilin interactions.

Cortical neurons communicate with various cortical and subcortical targets by way of stereotyped axon projections through the white matter. Slice overlay experiments indicate that the initial growth of cortical axons toward the white matter is regulated by a diffusible chemorepulsive signal localized near the marginal zone. Semaphorin III is a major component of this diffusible signal, and cortical neurons transduce this signal by way of the neuropilin-1 receptor. These observations indicate that semaphorin-neuropilin interactions play a critical role in the initial patterning of projections in the developing cortex.

Identification and characterization of novel substrates of Trk receptors in developing neurons.

Neurotrophins influence growth and survival of specific populations of neurons through activation of Trks, members of the receptor tyrosine kinase (RTK) family. In this report, we describe the identification and characterization of two substrates of Trk kinases, rAPS and SH2-B, which are closely related Src homolog 2 (SH2) domain-containing signaling molecules. rAPS and SH2-B are substrates of TrkB and TrkC in cortical neurons and SH2-B is a substrate of TrkA in sympathetic neurons. Moreover, rAPS and SH2-B bind to Grb2, and both are sufficient to mediate NGF induction of Ras, MAP kinase (MAPK), and morphological differentiation of PC12 cells. Lastly, antibody perturbation and transient transfection experiments indicate that SH2-B, or a closely related molecule, is necessary for NGF-dependent signaling in neonatal sympathetic neurons. Together, these observations indicate that rAPS and SH2-B mediate Trk signaling in developing neurons.

Neuropilin-2 is a receptor for semaphorin IV: insight into the structural basis of receptor function and specificity.

Neuropilins bind secreted members of the semaphorin family of proteins. Neuropilin-1 is a receptor for Sema III. Here, we show that neuropilin-2 is a receptor for the secreted semaphorin Sema IV and acts selectively to mediate repulsive guidance events in discrete populations of neurons. neuropilin-2 and semaIV are expressed in strikingly complementary patterns during neurodevelopment. The extracellular complement-binding (CUB) and coagulation factor domains of neuropilin-2 confer specificity to the Sema IV repulsive response, and these domains of neuropilin-1 are necessary and sufficient for binding of the Sema III semaphorin (sema) domain. The coagulation factor domains alone are necessary and sufficient for binding of the Sema III immunoglobulin- (Ig-) basic domain and the unrelated ligand, vascular endothelial growth factor (VEGF). Lastly, neuropilin-1 can homomultimerize and form heteromultimers with neuropilin-2. These results provide insight into how interactions between neuropilins and secreted semaphorins function to coordinate repulsive axon guidance during neurodevelopment.

Effects of PS1 deficiency on membrane protein trafficking in neurons.

We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.

A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos.

Several studies have characterized the upstream regulatory region of c-fos, and identified cis-acting elements termed the cyclic AMP (cAMP) response elements (CREs) that are critical for c-fos transcription in response to a variety of extracellular stimuli. Although several transcription factors can bind to CREs in vitro, the identity of the transcription factor(s) that activates the c-fos promoter via the CRE in vivo remains unclear. To help identify the trans-acting factors that regulate stimulus-dependent transcription of c-fos via the CREs, dominant-negative (D-N) inhibitor proteins that function by preventing DNA binding of B-ZIP proteins in a dimerization domain-dependent fashion were developed. A D-N inhibitor of CREB, termed A-CREB, was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper domain. The acidic extension of A-CREB interacts with the basic region of CREB forming a coiled-coil extension of the leucine zipper and thus prevents the basic region of wild-type CREB from binding to DNA. Other D-N inhibitors generated in a similar manner with the dimerization domains of Fos, Jun, C/EBP, ATF-2, or VBP did not block CREB DNA binding activity, nor did they inhibit transcriptional activation of a minimal promoter containing a single CRE in PC12 cells. A-CREB inhibited activation of CRE-mediated transcription evoked by three distinct stimuli: forskolin, which increases intracellular cAMP; membrane depolarization, which promotes Ca2+ influx; and nerve growth factor (NGF). A-CREB completely inhibited cAMP-mediated, but only partially inhibited Ca2+- and NGF-mediated, transcription of a reporter gene containing 750 bp of the native c-fos promoter. Moreover, glutamate induction of c-fos expression in primary cortical neurons was dependent on CREB. In contrast, induction of c-fos transcription by UV light was not inhibited by A-CREB. Lastly, A-CREB attenuated NGF induction of morphological differentiation in PC12 cells. These results suggest that CREB or its closely related family members are general mediators of stimulus-dependent transcription of c-fos and are required for at least some of the long-term actions of NGF.

Neuropilin is a semaphorin III receptor.

The semaphorin family contains a large number of phylogenetically conserved proteins and includes several members that have been shown to function in repulsive axon guidance. Semaphorin III (Sema III) is a secreted protein that in vitro causes neuronal growth cone collapse and chemorepulsion of neurites, and in vivo is required for correct sensory afferent innervation and other aspects of development. The mechanism of Sema III function, however, is unknown. Here, we report that neuropilin, a type I transmembrane protein implicated in aspects of neurodevelopment, is a Sema III receptor. We also describe the identification of neuropilin-2, a related neuropilin family member, and show that neuropilin and neuropilin-2 are expressed in overlapping, yet distinct, populations of neurons in the rat embryonic nervous system.

An NGF-TrkA-mediated retrograde signal to transcription factor CREB in sympathetic neurons.

Nerve growth factor (NGF) is a neurotrophic factor secreted by cells that are the targets of innervation of sympathetic and some sensory neurons. However, the mechanism by which the NGF signal is propagated from the axon terminal to the cell body, which can be more than 1 meter away, to influence biochemical events critical for growth and survival of neurons has remained unclear. An NGF-mediated signal transmitted from the terminals and distal axons of cultured rat sympathetic neurons to their nuclei regulated phosphorylation of the transcription factor CREB (cyclic adenosine monophosphate response element-binding protein). Internalization of NGF and its receptor tyrosine kinase TrkA, and their transport to the cell body, were required for transmission of this signal. The tyrosine kinase activity of TrkA was required to maintain it in an autophosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus, an NGF-TrkA complex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons.

Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase.

A signaling pathway has been elucidated whereby growth factors activate the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB), a critical regulator of immediate early gene transcription. Growth factor-stimulated CREB phosphorylation at serine-133 is mediated by the RAS-mitogen-activated protein kinase (MAPK) pathway. MAPK activates CREB kinase, which in turn phosphorylates and activates CREB. Purification, sequencing, and biochemical characterization of CREB kinase revealed that it is identical to a member of the pp90(RSK) family, RSK2. RSK2 was shown to mediate growth factor induction of CREB serine-133 phosphorylation both in vitro and in vivo. These findings identify a cellular function for RSK2 and define a mechanism whereby growth factor signals mediated by RAS and MAPK are transmitted to the nucleus to activate gene expression.