Retinoic acid stimulates the differentiation of PC12 cells that are deficient in cAMP-dependent protein kinase.

Retinoic acid (RA) induced neuronal differentiation in A126-1B2 cells and 123.7 cells, two mutant lines of PC12 that are deficient in cAMP-dependent protein kinase, but not in the parental PC12 cell line. A single exposure to RA was sufficient to cause neurite formation and inhibit cell division for a period of greater than 3 wk, suggesting that RA may cause a long-term, stable change in the state of these cells. In A126-1B2 cells, RA also induced the expression of other markers of differentiation including acetylcholinesterase and the mRNAs for neurofilament (NF-M) and GAP-43 as effectively as nerve growth factor (NGF). Neither NGF nor RA stimulated an increase in the expression of smg-25A in A126-1B2 cells, suggesting that the cAMP-dependent protein kinases may be required for an increase in the expression of this marker. RA also caused a rapid increase in the expression of the early response gene, c-fos, but did not effect the expression of egr-1. RA equivalently inhibited the division of A126-1B2 cells, 123.7 cells and parental PC12 cells, so RA induced differentiation is not an indirect response to growth arrest. In contrast, the levels of retinoic acid receptors (RAR alpha and RAR beta), and retinoic acid binding protein (CRABP) mRNA were strikingly higher in both A126-1B2 cells and 123.7 cells than in the parental PC12 cells. The deficiencies in cAMP-dependent protein kinase may increase the expression of CRABP and the RARs; and, thus, cAMP may indirectly regulate the ability of RA to control neurite formation and neural differentiation. Thus, RA appears to regulate division and differentiation of PC12 cells by a biochemical mechanism that is quite distinct from those used by peptide growth factors.

The activity of cAMP-dependent protein kinase is required at a posttranslational level for induction of voltage-dependent sodium channels by peptide growth factors in PC12 cells.

The synthesis and expression of voltage-dependent sodium (Na) channels is a crucial aspect of neuronal differentiation because of the central role these ion channels play in the generation of action potentials and the transfer of information in the nervous system. We have used rat pheochromocytoma (PC12) cell lines deficient in cAMP-dependent protein kinase (PKA) activity to examine the role of PKA in the induction of Na channel expression by nerve growth factor (NGF) and basic FGF (bFGF). In the parental PC12 cell line both NGF and bFGF elicit an increase in the density of functional Na channels, as determined from whole-cell patch clamp recordings. This increase does not occur in two PC12 cell lines deficient in both isozymes of PKA (PKAI and PKAII), and is strongly reduced in a third line deficient in PKAII, but not PKAI. Despite the inability of the neurotrophic factors to induce functional Na channel expression in the PKA-deficient cells, Northern blot hybridization studies and saxitoxin binding assays of intact cells indicate that NGF and bFGF are still capable of eliciting increases in both Na channel mRNA and Na channel protein in the membrane. Thus, PKA activity appears to be necessary at a posttranslational step in the synthesis and expression of functional Na channels, and thereby plays an important role in determining neuronal excitability.

Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways.

Calcium ions (Ca2+) act as an intracellular second messenger and can enter neurons through various ion channels. Influx of Ca2+ through distinct types of Ca2+ channels may differentially activate biochemical processes. N-Methyl-D-aspartate (NMDA) receptors and L-type Ca2+ channels, two major sites of Ca2+ entry into hippocampal neurons, were found to transmit signals to the nucleus and regulated gene transcription through two distinct Ca2+ signaling pathways. Activation of the multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaM kinase) was evoked by stimulation of either NMDA receptors or L-type Ca2+ channels; however, activation of CaM kinase appeared to be critical only for propagating the L-type Ca2+ channel signal to the nucleus. Also, the NMDA receptor and L-type Ca2+ channel pathways activated transcription by means of different cis-acting regulatory elements in the c-fos promoter. These results indicate that Ca2+, depending on its mode of entry into neurons, can activate two distinct signaling pathways. Differential signal processing may provide a mechanism by which Ca2+ controls diverse cellular functions.

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.

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.

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.

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.

Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock.

Mammalian circadian rhythms are regulated by a pacemaker within the suprachiasmatic nuclei (SCN) of the hypothalamus. The molecular mechanisms controlling the synchronization of the circadian pacemaker are unknown; however, immediate early gene (IEG) expression in the SCN is tightly correlated with entrainment of SCN-regulated rhythms. Antibodies were isolated that recognize the activated, phosphorylated form of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB). Within minutes after exposure of hamsters to light, CREB in the SCN became phosphorylated on the transcriptional regulatory site, Ser133. CREB phosphorylation was dependent on circadian time: CREB became phosphorylated only at times during the circadian cycle when light induced IEG expression and caused phase shifts of circadian rhythms. These results implicate CREB in neuronal signaling in the hypothalamus and suggest that circadian clock gating of light-regulated molecular responses in the SCN occurs upstream of phosphorylation of CREB.

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.

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.

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.

Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras.

A pathway by which calcium influx through voltage-sensitive calcium channels leads to mitogen-activated protein kinase (MAPK) activation has been characterized. In PC12 cells, membrane depolarization leading to calcium influx through L-type calcium channels activates the dual specificity MAPK kinase MEK1, which phosphorylates and activates MAPK. Calcium influx leads within 30 s to activation of the small guanine nucleotide-binding protein Ras. Moreover, activation of MAPK in response to calcium influx is inhibited by the dominant negative mutant RasAsn17, indicating that Ras activity is required for calcium signaling to MAPK. Ras is also activated by release of calcium from intracellular stores and by membrane depolarization of primary cortical neurons. The pleiotropic regulatory potential of both Ras and the MAPK pathway suggests that they may be central mediators of calcium signaling in the nervous system.

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.

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.

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.

Calcium activates serum response factor-dependent transcription by a Ras- and Elk-1-independent mechanism that involves a Ca2+/calmodulin-dependent kinase.

Enhanced levels of cytoplasmic Ca2+ due to membrane depolarization with elevated levels of KCl or exposure to the Ca2+ ionophore ionomycin stimulate serum response element (SRE)-dependent transcription in the pheochromocytoma cell line PC12. By using altered binding specificity mutants of transcription factors that bind to the SRE, it was demonstrated that in contrast to treatment with purified growth factors, such as nerve growth factor, the serum response factor (SRF), but not Elk-1, mediates Ca(2+)-regulated SRE-dependent transcription. Enhanced levels of cytoplasmic Ca2+ were found to trigger SRE-dependent transcription via a Ras-independent signaling pathway that appears to involve a Ca2+/calmodulin-dependent kinase (CaMK). Overexpression of a constitutively active form of CaMKIV stimulated SRF-dependent transcription. Taken together, these findings indicate that SRF is a versatile transcription factor that, when bound to the SRE, can function by distinct mechanisms and can mediate transcriptional responses to both CaMK- and Ras-dependent signaling pathways.

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.

A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity.

A signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.

Trans-synaptic regulation of gene expression.

Neurotransmitters regulate gene expression through second messenger cascades that transmit the signal from the plasma membrane to the nucleus of the postsynaptic cell. Ca2+ and cAMP are two of the second messengers that regulate gene expression in response to neurotransmitters. The Ca2+ and cAMP signals induce expression of a class of genes, termed immediate early genes, within minutes of neurotransmitter receptor activation. Many of these genes encode transcription factors that regulate the expression of late response genes. The results of recent experiments have elucidated mechanisms by which neurotransmitter-induced Ca2+ and cAMP signals regulate immediate early gene expression.