Neuronal calcium activates a Rap1 and B-Raf signaling pathway via the cyclic adenosine monophosphate-dependent protein kinase.

Activity-dependent regulation of neuronal events such as cell survival and synaptic plasticity is controlled by increases in neuronal calcium levels. These actions often involve stimulation of intracellular kinase signaling pathways. For example, the mitogen-activated protein kinase, or extracellular signal-regulated kinase (ERK), signaling cascade has increasingly been shown to be important for the induction of gene expression and long term potentiation. However, the mechanisms leading to ERK activation by neuronal calcium are still unclear. In the present study, we describe a protein kinase A (PKA)-dependent signaling pathway that may link neuronal calcium influx to ERKs via the small G-protein, Rap1, and the neuronal Raf isoform, B-Raf. Thus, in PC12 cells, depolarization-mediated calcium influx led to the activation of B-Raf, but not Raf-1, via PKA. Furthermore, depolarization also induced the PKA-dependent stimulation of Rap1 and led to the formation of a Rap1/B-Raf signaling complex. In contrast, depolarization did not lead to the association of Ras with B-Raf. The major action of PKA-dependent Rap1/B-Raf signaling in neuronal cells is the activation of ERKs. Thus, we further show that, in both PC12 cells and hippocampal neurons, depolarization-induced calcium influx stimulates ERK activity in a PKA-dependent manner. Given the fact that both Rap1 and B-Raf are highly expressed in the central nervous system, we suggest that this signaling pathway may regulate a number of activity-dependent neuronal functions.

Selective blockade of axonogenesis in cultured hippocampal neurons by the tyrosine phosphatase inhibitor orthovanadate.

Protein tyrosine phosphorylation has been implicated in several aspects of neurite outgrowth regulation. To address specific roles in early neuronal morphogenesis, hippocampal neurons in culture were treated with the tyrosine phosphatase inhibitor orthovanadate. This treatment completely suppressed axon formation, yet enhanced formation of minor neurites. The inhibition of axonogenesis was dose dependent and occurred in parallel with a marked increase in cellular phosphotyrosine immunoreactivity, which was especially concentrated within neuritic growth cones and showed partial colocalization with f-actin. Both the blockade of axonogenesis and the elevation of phosphotyrosine were completely reversible. An additional and unexpected effect of orthovanadate was the appearance of many binucleate neurons. Immunoblotting experiments using a phosphotyrosine-specific antibody revealed an orthovanadate-induced reversible hyperphosphorylation of several protein bands, especially of two at 115 and 125 kD. These data suggest a potentially important role for tyrosine phosphatases and their phosphoprotein substrates in axonogenesis.

Delayed localization of synelfin (synuclein, NACP) to presynaptic terminals in cultured rat hippocampal neurons.

Synelfin is a presynaptic protein of unknown function that is differentially regulated in the avian song control circuit during the critical period for song learning; in humans, it gives rise to an amyloidogenic peptide found in senile plaques of Alzheimer's disease. To gain insight into the potential involvement of synelfin in synapse development, we investigated its expression in neurons cultured from the embryonic rat hippocampus. These neurons express a variety of defined synaptic proteins, and form numerous synaptic connections after several days in culture. Synapsin I, a synaptic vesicle-associated protein, was detected within one day after the neurons were put in culture, but significant immunoreactivity for synelfin was not detected until approximately 5 days in vitro (DIV). By 3 DIV, synapsin-positive puncta (previously shown to correspond to presynaptic specializations) were detected surrounding the soma and proximal dendritic processes, whereas comparable aggregations of synelfin did not appear until several days later. By 14 DIV the punctate concentrations of synelfin and synapsin overlapped completely. Thus synelfin is expressed in these cultured neurons and eventually becomes localized to presynaptic terminals, but it is absent from these specializations when they first form. We conclude that presynaptic terminals can change in molecular composition, and that synelfin is associated with later stages in synaptic development or modulation.

Low density lipoprotein receptor-related protein is expressed early and becomes restricted to a somatodendritic domain during neuronal differentiation in culture.

Low density lipoprotein receptor-related protein (LRP) is a multi-functional receptor which mediates the endocytotic uptake of several ligands implicated in neuronal pathophysiology. In this study, LRP expression and localization, in cultured hippocampal neurons from 18-day-old rats, were examined by immunofluorescence microscopy. LRP was restricted to the cell bodies and dendrites of mature neurons, where it was uniformly distributed on both dendritic shafts and spines. Immunoreactive protein was detected within the first 24 h of culture and acquired a polarized distribution by the end of the first week. Expression of LRP mRNA by the cultured neurons was demonstrated by Northern blot analysis. Binding studies with the LRP ligand, activated alpha2-macroglobulin, confirmed that LRP was present and functional on the hippocampal neuron cell surface. These studies demonstrate that neuronal LRP undergoes selective compartmentation during neuronal maturation and suggest that LRP-mediated endocytosis is largely restricted to the somatodendritic compartment.

A spatial gradient of tau protein phosphorylation in nascent axons.

Mechanisms underlying axonogenesis remain obscure. Although a large number of proteins eventually become polarized to the axonal domain, in no case does protein compartmentalization occur before or simultaneous with the earliest morphological expression of axonal properties. How then might initially unpolarized proteins, such as the microtubule-associated protein tau, play a role in the microdifferentiation of axons? We hypothesized that tau function could be locally regulated by phosphorylation during the period of axonogenesis. To test this hypothesis, we mapped relative levels of tau phosphorylation within developing cultured hippocampal neurons. This was accomplished using calibrated immunofluorescence ratio measurements employing phosphorylation state-dependent and state-independent antibodies. Tau in the nascent axon is more highly dephosphorylated at the site recognized by the tau-1 antibody than tau in the somatodendritic compartment. The change in phosphorylation state from soma to axon takes the form of a smooth proximo-distal gradient, with tau in the soma, immature dendrites and proximal axon approximately 80% phosphorylated at the tau-1 site, and that in the axonal growth cone only 20% phosphorylated. The existence of real spatial differences in tau phosphorylation state was confirmed by in situ phosphatase and kinase treatment. Pervanadate, a tyrosine phosphatase inhibitor, induced rapid tau dephosphorylation within live cells, effectively abolishing the phosphorylation gradient. Thus, the gradient is dynamic and potentially regulatable by upstream signals involving tyrosine phosphorylation. Phosphorylation gradients are likely to be present on many neuronal proteins in addition to tau, and their modulation by transmembrane signals could direct the establishment of polarity.

The economics of neurite outgrowth--the addition of new membrane to growing axons.

Recent studies have shown that axonal growth is disrupted by treatments that block the synthesis of membrane components or their delivery by microtubule-based transport. This implies that a continuous supply of newly synthesized membrane components is necessary to sustain growth. In contrast, no clear consensus has yet been achieved about the site of insertion of new membrane components in the membrane of the growing axon, despite the application of new and refined biophysical and molecular techniques to the study of this issue. Until the site of insertion of new membrane components is resolved, little progress can be made in defining the feedback mechanisms by which the supply of new membrane components is co-ordinated with the demands of growth, particularly in cases where the dynamics of neurite growth change from minute to minute.

Microtubule-associated proteins, phosphorylation gradients, and the establishment of neuronal polarity.

Axonogenesis is the earliest step in acquisition of neuronal polarity. The subcellular mechanisms underlying this pivotal event are unknown. Because of the abundant presence and functional necessity of microtubule-associated proteins in growing neurites, a large effort has been directed at characterizing their role in establishment and maintenance of neuronal polarity. One unsolved puzzle is how MAPs, most of which are unpolarized in early stages of development, can locally influence microdifferentiation of axons and dendrites. In this review, we discuss recent evidence suggesting that locally controlled phosphorylation of microtubule-associated proteins tau and MAP1B may play a role in establishment of polarity and early axonal outgrowth.

The microtubule cytoskeleton and the development of neuronal polarity.

The concept that axons and dendrites represent a fundamental polarization of the nerve cell has been borne out by numerous morphological, functional, and molecular studies. How does polarity arise during development? We and others have focused on the role of the microtubule cytoskeleton because microtubules (a) are essential components of axons and dendrites; (b) possess an inherent polarity at the molecular level; (c) are regulated by interactions with microtubule associated proteins (MAPs), some of which have polarized distributions in mature neurons. Here we review data on the initial acquisition of polarity as observed in neuronal culture and roles for microtubules and MAPs in this morphogenetic event. We present data clarifying some previously conflicting results on tau localization during the establishment of polarity and provide new evidence that phosphorylation of tau is spatially regulated during the development of polarity in culture. Elucidation of mechanisms locally regulating tau phosphorylation during normal neuronal development may provide clues to the significance of its abnormal phosphorylation in Alzheimer's disease.

An electron microscopic analysis of hippocampal neurons developing in culture: early stages in the emergence of polarity.

In culture, hippocampal neurons initially establish several short, minor processes. The initial step in the emergence of polarity is marked by the rapid and selective growth of one of these processes, which becomes the axon. Subsequently the remaining processes become dendrites. We examined the ultrastructure of hippocampal neurons before and after the emergence of the axon. The minor processes in cells that had not yet formed axons were somewhat variable in appearance, but we found no ultrastructural feature that indicated which minor process might become the axon. The emergence of the axon was marked by several changes in its ultrastructure. The axon contained a sevenfold lower density of polyribosomes than the minor processes. In addition, axonal growth cones contained a pronounced concentration of membranous elements that resembled endoplasmic reticulum, elements that were rare in the growth cones of minor processes. Axons and minor processes did not differ in microtubule density. In order to gauge how rapidly these ultrastructural changes occur, we examined cells with short axons that, from their length, were estimated to have emerged only hours earlier. The preferential exclusion of polyribosomes from the axon and the concentration of reticular membrane in the axonal growth cone were already evident in such cells. These observations demonstrate that exclusion of ribosomes from the axon occurs early in development, about as soon as the axon can be identified. In contrast, previous work has shown that the differences in microtubule polarity orientation that distinguish mature axons and dendrites, and that have been proposed to account for the selective segregation of some constituents in neurons, first appear at a later stage of development (Baas et al., 1989). These observations also demonstrate that the accumulation of reticular membrane elements in growth cones, which has been noted previously, occurs preferentially in axonal growth cones and is closely correlated in time with the initial specification of the axon. The selective concentration of these elements in axonal growth cones could be associated with the uniquely rapid rate of axonal growth.

Laminin selectively enhances axonal growth and accelerates the development of polarity by hippocampal neurons in culture.

We have examined the effects of laminin on the morphological development of embryonic rat hippocampal neurons maintained in tissue culture. Forty-eight hours after plating, neurons grown on a polylysine-coated substrate had become polarized, typically having one long axon and 4 or 5 minor processes. Adsorption of laminin to the substrate did not cause changes in the number of axons extended by hippocampal neurons but did cause significant increases in the length of the axonal plexus and in axonal branching. In contrast to its effects on axons, laminin did not influence the number, length, or branching of the minor processes that eventually become dendrites or the morphology of definite dendrites as assessed after 7 days in culture. In addition to selectively enhancing axonal growth, laminin greatly increased the rate of polarization of hippocampal neurons such that most became polarized within 18 h. Analysis of the time course of laminin's effects revealed that the acceleration of polarization was not associated with a change in the time of initial process formation, but rather with a selective stimulation of the growth of the longest process at all times from the 12th through the 48th h in vitro. These data suggest that even though the basic shape of hippocampal neurons may be intrinsically programmed, critical aspects of their morphological development may be modulated by extracellular matrix molecules such as laminin.

Getting the message from the gene to the synapse: sorting and intracellular transport of RNA in neurons.

A key question in cellular neurobiology is how neurons target molecules to cellular microdomains at a distance from the nucleus. Of special importance are the thousands of postsynaptic sites that form the basis for synaptic communication. Recent evidence suggests that an important aspect of molecular trafficking involves differential sorting, selective intracellular transport, and docking of particular mRNA molecules and associated protein synthetic machinery at postsynaptic sites. This offers the potential for local regulation of the production of key proteins in response to conditions at individual synapses. This article reviews what is known about the mechanisms of mRNA trafficking in neurons and in other cells ranging from oocytes to oligodendrocytes, and considers the possible role that mRNA trafficking and the resulting local synthesis of particular proteins may play in cellular function.

Dendritic transport: quantitative analysis of the time course of somatodendritic transport of recently synthesized RNA.

We have previously reported that recently synthesized RNA is selectively transported into the dendrites of hippocampal neurons grown in culture (Davis et al., 1987). The present study provides further details about this transport process, focusing especially on the velocity of transport, by comparing the velocity of dendritic transport of RNA in neurons of different ages and in the branched and unbranched dendrites of individual neurons. In our previous study, we recognized that calculations of transport velocity could be compromised because transport was being evaluated in a population of dendrites of varying lengths. The present study uses a mathematical modeling approach to determine how the morphology of the population of dendrites would affect the analysis of transport velocity. Focusing first on a simple model, we compared the distribution of transported material at various times when all dendrites were of the same length and when the population included dendrites of different lengths. We found that the distance of labeling increased linearly over time when all dendrites were of the same length, but increased with a negatively accelerating curve when dendrites were of different lengths. We then determined the actual distribution of dendritic lengths in cultured hippocampal neurons, based on immunostaining with an antibody directed against the selective dendritic marker, microtubule-associated protein 2 (MAP2). Using a computer model, we calculated the mean distance of transport as a function of time in this population of dendrites, assuming different velocities of transport. The velocity that best fit the measured distances of RNA transport in both 7- and 15-d-old neurons was 11 microns/hr (0.26 mm/d). However, for the dendrites exhibiting the longest distance of labeling, the best-fitting curve assumed a velocity of 21 microns/hr in both 7- and 15-d-old neurons (0.50 mm/d). Comparisons of transport in branched and unbranched dendrites revealed that the distance of labeling over branched dendrites was consistently longer than over unbranched dendrites of individual neurons. However, neurons with a larger proportion of branched dendrites did not exhibit a greater mean distance of transport. The density of silver grains was higher over branched than over unbranched dendrites, suggesting that a greater amount of recently synthesized RNA may be transported into branched dendrites. Taken together, these results suggest that RNA transport into dendrites is regulated differentially in the dendrites of individual neurons.

The establishment of polarity by hippocampal neurons: the relationship between the stage of a cell's development in situ and its subsequent development in culture.

Neurons removed from the embryonic hippocampus and placed into culture develop structurally and functionally distinct axonal and dendritic processes. The central issue addressed in this study concerns the extent to which the sequence of events which results in the differentiation of neurites by hippocampal neurons in culture is influenced by the cell's state of development in situ. [3H]thymidine was administered to pregnant rats either on Embryonic Day 15 (E15) or on E18.5 to label hippocampal neurons at known stages of their development. All fetuses were sacrificed on E19. Some of the fetal brains were sectioned and examined by autoradiography to determine the location of labeled cells in the hippocampus. The remaining brains were used to prepare hippocampal cell cultures. Neurons labeled at E18.5 remained confined to the ventricular zone at E19. Those labeled at E15 had completed their migration to the cortical plate. Other data suggest that the former cells had not yet initiated process outgrowth, while the latter cells had begun to elaborate both axons and dendrites. When introduced into culture, both populations of cells developed axons and dendrites and both compartmentalized MAP2 to the dendritic domain. Moreover, despite marked differences in their developmental state at the time of introduction into culture, both underwent the same sequence of developmental events leading to axonal and dendritic development. In a few cases cells that incorporated [3H]thymidine in situ at E18.5 apparently underwent mitosis in culture. These neurons also developed axons and dendrites appropriately. These results indicate that hippocampal neurons become polarized in culture, even if they have never developed axons or dendrites in situ, and do so as efficiently as cells that have become polarized before being placed into culture. Moreover, they indicate that the same sequence of events leading to the establishment of polarity occurs for hippocampal neurons with different developmental histories prior to culturing.

Changes in microtubule polarity orientation during the development of hippocampal neurons in culture.

Microtubules in the dendrites of cultured hippocampal neurons are of nonuniform polarity orientation. About half of the microtubules have their plus ends oriented distal to the cell body, and the other half have their minus ends distal; in contrast, microtubules in the axon are of uniform polarity orientation, all having their plus ends distal (Baas, P.W., J.S. Deitch, M. M. Black, and G. A. Banker. 1988. Proc. Natl. Acad. Sci. USA. 85:8335-8339). Here we describe the developmental changes that give rise to the distinct microtubule patterns of axons and dendrites. Cultured hippocampal neurons initially extend several short processes, any one of which can apparently become the axon (Dotti, C. G., and G. A. Banker. 1987. Nature [Lond.]. 330:477-479). A few days after the axon has begun its rapid growth, the remaining processes differentiate into dendrites (Dotti, C. G., C. A. Sullivan, and G. A. Banker. 1988. J. Neurosci. 8:1454-1468). The polarity orientation of the microtubules in all of the initial processes is uniform, with plus ends distal to the cell body, even through most of these processes will become dendrites. This uniform microtubule polarity orientation is maintained in the axon at all stages of its growth. The polarity orientation of the microtubules in the other processes remains uniform until they begin to grow and acquire the morphological characteristics of dendrites. It is during this period that microtubules with minus ends distal to the cell body first appear in these processes. The proportion of minus end-distal microtubules gradually increases until, by 7 d in culture, about equal numbers of dendritic microtubules are oriented in each direction. Thus, the establishment of regional differences in microtubule polarity orientation occurs after the initial polarization of the neuron and is temporally correlated with the differentiation of the dendrites.

Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite.

We have analyzed the polarity orientation of microtubules in the axons and dendrites of cultured rat hippocampal neurons. As previously reported of axons from other neurons, microtubules in these axons are uniform with respect to polarity; (+)-ends are directed away from the cell body toward the growth cone. In sharp contrast, microtubules in the mid-region of the dendrite, approximately 75 microns from the cell body, are not of uniform polarity orientation. Roughly equal proportions of these microtubules are oriented with (+)-ends directed toward the growth cone and (+)-ends directed toward the cell body. At distances within 15 micron of the growth cone, however, microtubule polarity orientation in dendrites is similar to that in axons; (+)-ends are uniformly directed toward the growth cone. These findings indicate a clear difference between axons and dendrites with respect to microtubule organization, a difference that may underlie the differential distribution of organelles within the neuron.

The establishment of polarity by hippocampal neurons in culture.

By the end of the first week in culture, hippocampal neurons have established a single axon and several dendrites. These 2 classes of processes differ in their morphology, in their molecular composition, and in their synaptic polarity (Bartlett and Banker, 1984a, b; Caceres et al., 1984). We examined the events during the first week in culture that lead to the establishment of this characteristic form. Hippocampal cells were obtained from 18 d fetal rats, plated onto polylysine-treated coverslips, and maintained in a serum-free medium. The development of individual cells was followed by sequential photography at daily intervals until both axons and dendrites had been established; identification of the processes was confirmed by immunostaining for MAP2, a dendritic marker. Time-lapse video recording was used to follow the early stages of process formation. Hippocampal neurons acquired their characteristic form by a stereotyped sequence of developmental events. The cells first established several, apparently identical, short processes. After several hours, one of the short processes began to grow very rapidly; it became the axon. The remaining processes began to elongate a few days later and grew at a much slower rate. They became the cell's dendrites. Neurons that arose following mitosis in culture underwent this same sequence of developmental events. In a few instances, 2 processes from a cell exhibited the rapid growth typical of axons, but only one maintained this growth; the other retracted and became a dendrite. Axons branched primarily by the formation of collaterals, not by bifurcation of growth cones. As judged by light microscopy, processes are not specified as axons or dendrites when they arise. The first manifestation of neuronal polarity is the acquisition of axonal characteristics by one of the initial processes; subsequently the remaining processes become dendrites.

Selective dendritic transport of RNA in hippocampal neurons in culture.

Typical neurons of the central nervous system (CNS) elaborate tens of thousands of membrane specializations at sites of synaptic contacts on their dendrites. To construct, maintain, and modify these specializations, neurons must produce and deliver the appropriate molecular constituents to particular synaptic sites. Previous studies have revealed that polyribosomes are selectively positioned beneath postsynaptic sites, suggesting that in neurons, as in other cell types, protein synthetic machinery is located at or near the sites where particular proteins are needed. The mechanisms that deliver ribosomes and messenger RNA to their specific destinations in cells are therefore of considerable interest. Here we describe a system for RNA transport in dendrites that could provide a mechanism for the delivery of ribosomes and mRNA to synaptic sites in dendrites. Hippocampal neurons grown in culture incorporate 3H-uridine in the nucleus, then selectively transport the newly synthesized RNA into dendrites at a rate of about 0.5 mm day-1. The transport is inhibited by metabolic poisons, suggesting that it is an active, energy-dependent process. The RNA may be transported in association with the cytoskeleton.

Experimentally induced alteration in the polarity of developing neurons.

Despite the great diversity of shapes exhibited by different classes of nerve cells, nearly all neurons share one feature in that they have a single axon and several dendrites. The two types of processes differ in their morphology, in their rate of growth, in the macromolecular composition of their cytoskeletons and surface membranes, and in their synaptic polarity. When hippocampal neurons are dissociated from the embryonic brain and cultured, they reproducibly establish this basic form with a single axon and several dendrites, despite the absence of any spatially organized environmental cues, and without the need for cell to cell contact. We have cut the axons of young hippocampal neurons within a day of their development: in some cases the initial axon regenerated, but more frequently one of the other processes, which if undisturbed would have become a dendrite, instead became the axon. Frequently the stump of the original axon persisted following the transection and subsequently became a dendrite. Evidently the neuronal processes that first develop in culture have the capacity to form either axons or dendrites. The acquisition of axonal characteristics by one neuronal process apparently inhibits the others from becoming axons, so they subsequently become dendrites.

The expression and distribution of the microtubule-associated proteins tau and microtubule-associated protein 2 in hippocampal neurons in the rat in situ and in cell culture.

Using a monoclonal antibody against the microtubule-associated protein tau we compared the distribution and the biochemical maturation of this protein in hippocampal pyramidal neurons in the rat in tau and in culture. In tissue sections from mature animals tau was localized heterogeneously within neurons. It was concentrated in axons; dendrites and somata showed little or no staining. In hippocampal cultures ranging from 12 h to 4 weeks in vitro tau was present in neurons but not in glial cells, as it is in situ. Within cultured neurons, however, tau was not compartmentalized but was present throughout the dendrites, axons and somata. Immunoblotting experiments showed that the biochemical maturation of tau that occurs in situ also failed to occur in culture. The young form of tau persisted, and the adult forms did not develop. In contrast the biochemical maturation and the compartmentalization of microtubule-associated protein 2 occurred normally in hippocampal cultures. These results show that the biochemical maturation and the intraneuronal compartmentalization of these two microtubule-associated proteins are independently controlled. Despite the non-restricted distribution of tau in hippocampal neurons in culture, and despite the presence of only the immature isoform which has a lessened stimulatory effect on microtubule polymerization, axons and dendrites appear to grow normally and to exhibit appropriate functional properties.

Immunocytochemical localization of tubulin and microtubule-associated protein 2 during the development of hippocampal neurons in culture.

In dissociated-cell cultures prepared from the embryonic rat hippocampus, neurons establish both axons and dendrites, which differ in geometry, in ultrastructure, and in synaptic polarity. We have used immunocytochemistry with monoclonal antibodies to study the regional distribution of beta-tubulin and micro-tubule-associated protein 2 (MAP2) in hippocampal cultures and their localization during early stages of axonal and dendritic development. After development for a week or more in culture, when axons and dendrites were well-differentiated, the distribution of these two proteins was quite different. Beta-tubulin was present throughout the nerve cell, in soma, dendrites, and axon. It was also present in all classes of non-neuronal cells, astrocytes, fibroblasts, and a presumptive glial progenitor cell. In contrast, MAP2 was preferentially localized to nerve cells; within neurons, MAP2 was present in soma and dendrites, but little or no immunostaining was detectable in axons. Both beta-tubulin and MAP2 were present in nerve cells at the time of plating. From the earliest stages of process extension, beta-tubulin was present in all neuronal processes, both axons and dendrites. Surprisingly, MAP2 was also initially present in both axons and dendrites, extending as far as the axonal growth cone. With subsequent development, MAP2 staining was selectively lost from the axon so that after 1 week in vitro little or no axonal staining remained. Taken together with earlier results (Cáceres et al., 1984a), these data indicate that the establishment of neuronal polarity, as manifested by the molecular differentiation of the axonal and dendritic cytoskeleton, occurs largely under endogenous control, even under culture conditions in which cell interactions are greatly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)