The role of a LAR-like receptor tyrosine phosphatase in growth cone collapse and mutual-avoidance by sibling processes

Among the many cells or parts of cells that a growth cone may encounter during its embryonic migrations are other processes or parts of its parent cell. Such an event can be expected to be relatively frequent in the genesis of neuronal arbors, for instance, where the density of innervation of a target region can be quite high. Few experimental studies have addressed the very interesting question of whether a process "recognizes" siblings in some unique way, in a manner that can be distinguished from, say, how it interacts with unrelated cells. One example can be found in the leech, where sibling branches in the terminal fields of identified mechanosensory cells avoid each other strictly while permitting some significant continuing contact and overlap with homologues, a phenomenon that has been dubbed "self-avoidance." Another example has been reported in cultured Helisoma neurons, where severing a branch of a neuron allows sibling neurites to form electrical junctions with it, although normally sibling neurites do not do so. In both of these instances, coincidental activity was proposed as one means to achieve recognition of self and as possibly leading to the blocking of a continuing interaction among the parts, although alternative explanations were indeed considered possible.     

Steps in the formation of a bipolar outgrowth pattern by cultured neurons, and their substrate dependence.

We studied the steps in the formation of the bipolar outgrowth pattern of cultured adult Anterior Pagoda (AP) neurons of the leech growing on a central nervous system (CNS) homogenate as substrate. This pattern, which consists of two primary neurites directed in opposite directions plus some bifurcations, resembles their embryonic pattern but is different from the patterns they develop in culture on leech laminin or Concanavalin A as substrates. In eight neurons that were studied, one primary neurite formed and branched several hours before the second one. Time-lapse video analysis showed that between 12 and 36 h of growth, the more proximal branch of the early neurite migrated retrogradely, rotated, and formed the second primary branch. Both neurites elongated until the total neurite length reached 130-160 microm, when the elongation of primary neurites became synchronous with the retraction of secondary processes, suggesting competition. The substrate dependence of these events was tested by plating AP neurons on leech laminin. On this substrate AP neurons produced multiple independent primary neurites with branches. Retraction of some large branches was followed by their regrowth, and did not correlate with the changes in other neurites. We propose that the dynamics in the formation of the bipolar outgrowth pattern of AP neurons arise from inhibitory extracellular matrix molecules, which reduce the synthesis of precursors for neurite formation.     

Steps in the formation of a bipolar outgrowth pattern by cultured neurons,and their substrate dependence

We studied the steps in the formation of the bipolar outgrowth pattern of cultured adult Anterior Pagoda (AP) neurons of the leech growing on a central nervous system (CNS) homogenate as substrate. This pattern, which consists of two primary neurites directed in opposite directions plus some bifurcations, resembles their embryonic pattern but is different from the patterns they develop in culture on leech laminin or Concanavalin A as substrates. In eight neurons that were studied, one primary neurite formed and branched several hours before the second one. Time‐lapse video analysis showed that between 12 and 36 h of growth, the more proximal branch of the early neurite migrated retrogradely, rotated, and formed the second primary branch. Both neurites elongated until the total neurite length reached 130–160 μm, when the elongation of primary neurites became synchronous with the retraction of secondary processes, suggesting competition. The substrate dependence of these events was tested by plating AP neurons on leech laminin. On this substrate AP neurons produced multiple independent primary neurites with branches. Retraction of some large branches was followed by their regrowth, and did not correlate with the changes in other neurites. We propose that the dynamics in the formation of the bipolar outgrowth pattern of AP neurons arise from inhibitory extracellular matrix molecules, which reduce the synthesis of precursors for neurite formation. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 106–117, 2002; DOI 10.1002/neu.10017     

The possible role of "sibling neurite bias" in the coordination of neurite extension, branching, and survival

In this review we consider a novel mechanism, "sibling neurite bias," which may explain aspects of the coordination of elongation, branching, and resorption among different neurites growing from the same neuronal cell body. In this model, growing neurites which incorporate structural precursors at higher rates would deplete the cellular pool of precursors available to their "sibling" neurites; neurites would compete for survival, but in addition they would bias each other's behavior during active growth. Evidence is reviewed that "sibling neurite bias" may contribute to the establishment and stabilization of specific neural connections. Specific examples examined include the loss of polyinnervation at the developing neuromuscular junction, contextual mapping in the retino-tectal system, and selective neurite growth patterns and synaptic connections in nerve tissue culture model systems.     

The possible role of “sibling neurite bias” in the coordination of neurite extension,branching, and survival

In this review we consider a novel mechanism, “sibling neurite bias,” which may explain aspects of the coordination of elongation, branching, and resorption among different neurites growing from the same neuronal cell body. In this model, growing neurites which incorporate structural precursors at higher rates would deplete the cellular pool of precursors available to their “sibling” neurites; neurites would compete for survival, but in addition they would bias each other's behavior during active growth. Evidence is reviewed that “sibling neurite bias” may contribute to the establishment and stabilization of specific neural connections. Specific examples examined include the loss of polyinnervation at the developing neuromuscular junction, contextual mapping in the retino-tectal system, and selective neurite growth patterns and synaptic connections in nerve tissue culture model systems.     

Extracellular matrix molecules in development and regeneration of the leech CNS.

As neurons grow to their targets their processes elongate, branch and form specialized endings into which are inserted appropriate ion channels. Our aim has been to analyse the role of the extracellular matrix molecules laminin and tenascin in inducing growth and in determining the form and physiological properties of growing neurites. A preparation in which development and regeneration can be followed at the cellular and molecular level in the animal and in tissue culture is the central nervous system (CNS) of the leech. In leech extracellular matrix (ECM) both laminin and tenascin are present; the molecules are structurally similar but not identical to their vertebrate counterparts. Tenascin extracted from leech ECM shows a typical hexabrachial structure whereas laminin shows a typical cruciform structure in rotary shadowed preparations. Leech laminin purified by means of a monoclonal antibody is a molecule of about 1000 kDa, with a polypeptide composition of 340, 200, 180 and 160 kDa. Substrates that contain tenascin or laminin produce rapid and reliable outgrowth of neurites by identified cells. A remarkable finding is that the outgrowth pattern produced by an individual neuron depends in part on its identity, in part on the substrate upon which it is placed. For example, a Retzius cell grows in a quite different configuration and far more rapidly on laminin substrate than does another type of neuron containing the same transmitter (serotonin); and the pattern of outgrowth of the Retzius cell is different on laminin and on the plant lectin Con A (concanavalin A). Thus Con A induces the growth of processes that are shorter, thicker, more curved and contain fewer calcium channels than those grown on laminin. To determine whether laminin can also influence neurite outgrowth in the animal, immunocytological techniques have been used to follow its distribution in the extracellular matrix of normal, developing and regenerating leech CNS. In adult leeches neuronal processes in the CNS are not in contact with laminin which is confined to the surrounding extracellular matrix. In embryos however, laminin staining appears between ganglionic primordia along the pathways that neurons will follow. Similarly, after injury to the adult CNS, laminin accumulates at the very sites at which sprouting and regeneration begin. How the laminin becomes redistributed to appear in the region of injury has not yet been established. Together these findings suggest a key role for laminin and for other extracellular matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey

In larval lamprey, descending brain neurons, which regenerate their axons following spinal cord injury, were isolated and examined in cell culture to identify some of the factors that regulate neurite outgrowth. Focal application of 5 mM or 25 mM L-glutamate to single growth cones inhibited outgrowth of the treated neurite, but other neurites from the same neuron were not inhibited, an effect that has not been well studied for neurons in other systems. Glutamate-induced inhibition of neurite outgrowth was abolished by 10 mM kynurenic acid. Application of high potassium media to growth cones inhibited neurite outgrowth, an effect that was blocked by 2 mM cobalt or 100 microM cadmium, suggesting that calcium influx via voltage-gated channels contributes to glutamate-induced regulation of neurite outgrowth. Application of glutamate to growth cones in the presence of 2 microM omega-conotoxin MVIIC (CTX) still inhibited neurite outgrowth, while CTX blocked high potassium-induced inhibition of neurite outgrowth. Thus, CTX blocked virtually all of the calcium influx resulting from depolarization. To our knowledge, this is the first direct demonstration that calcium influx via ligand-gated ion channels can contribute to regulation of neurite outgrowth. Finally, focal application of glutamate to the cell bodies of descending brain neurons inhibited outgrowth of multiple neurites from the same neuron, and this is the first demonstration that multiple neurites can be regulated in this fashion. Signaling mechanisms involving intracellular calcium, similar to those shown here, may be important for regulating axonal regeneration following spinal cord injury in the lamprey.     

Complementary chimeric isoforms reveal Dscam1 binding specificity in vivo

Dscam1 potentially encodes 19,008 ectodomains of a cell recognition molecule of the immunoglobulin (Ig) superfamily through alternative splicing. Each ectodomain, comprising a unique combination of three variable (Ig) domains, exhibits isoform-specific homophilic binding in?vitro. Although we have proposed that the ability of Dscam1 isoforms to distinguish between one another is crucial for neural circuit assembly, via a process called self-avoidance, whether recognition specificity is essential in?vivo has not been addressed. Here we tackle this issue by assessing the function of Dscam1 isoforms with altered binding specificities. We generated pairs of chimeric isoforms that bind to each other (heterophilic) but not to themselves (homophilic). These isoforms failed to support self-avoidance or did so poorly. By contrast, coexpression of complementary isoforms within the same neuron restored self-avoidance. These data establish that recognition between Dscam1 isoforms on neurites of the same cell provides the molecular basis for self-avoidance.     

The Proprotein Convertase KPC-1/Furin Controls Branching and Self-avoidance of Sensory Dendrites in Caenorhabditis elegans

Animals sample their environment through sensory neurons with often elaborately branched endings named dendritic arbors. In a genetic screen for genes involved in the development of the highly arborized somatosensory PVD neuron in C. elegans, we have identified mutations in kpc-1, which encodes the homolog of the proprotein convertase furin. We show that kpc-1/furin is necessary to promote the formation of higher order dendritic branches in PVD and to ensure self-avoidance of sister branches, but is likely not required during maintenance of dendritic arbors. A reporter for kpc-1/furin is expressed in neurons (including PVD) and kpc-1/furin can function cell-autonomously in PVD neurons to control patterning of dendritic arbors. Moreover, we show that kpc-1/furin also regulates the development of other neurons in all major neuronal classes in C. elegans, including aspects of branching and extension of neurites as well as cell positioning. Our data suggest that these developmental functions require proteolytic activity of KPC-1/furin. Recently, the skin-derived MNR-1/menorin and the neural cell adhesion molecule SAX-7/L1CAM have been shown to act as a tripartite complex with the leucine rich transmembrane receptor DMA-1 on PVD mechanosensory to orchestrate the patterning of dendritic branches. Genetic analyses show that kpc-1/furin functions in a pathway with MNR-1/menorin, SAX-7/L1CAM and DMA-1 to control dendritic branch formation and extension of PVD neurons. We propose that KPC-1/furin acts in concert with the ‘menorin’ pathway to control branching and growth of somatosensory dendrites in PVD.     

Synaptic Integration in Electrically Coupled Neurons

Interactions among chemical and electrical synapses regulate the patterns of electrical activity of vertebrate and invertebrate neurons. In this investigation we studied how electrical coupling influences the integration of excitatory postsynaptic potentials (EPSPs). Pairs of Retzius neurons of the leech are coupled by a nonrectifying electrical synapse by which chemically induced synaptic currents flow from one neuron to the other. Results from electrophysiology and modeling suggest that chemical synaptic inputs are located on the coupled neurites, at 7.5 μm from the electrical synapses. We also showed that the space constant of the coupled neurites was 100 μm, approximately twice their length, allowing the efficient spread of synaptic currents all along both coupled neurites. Based on this cytoarchitecture, our main finding was that the degree of electrical coupling modulates the amplitude of EPSPs in the driving neurite by regulating the leak of synaptic current to the coupled neurite, so that the amplitude of EPSPs in the driving neurite was proportional to the value of the coupling resistance. In contrast, synaptic currents arriving at the coupled neurite through the electrical synapse produced EPSPs of constant amplitude. This was because the coupling resistance value had inverse effects on the amount of current arriving and on the impedance of the neurite. We propose that by modulating the amplitude of EPSPs, electrical synapses could regulate the firing frequency of neurons.     

Neurotoxicity caused by didanosine on cultured dorsal root ganglion neurons

The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites. ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities caused by ddI in both culture models may have a key role in neurite degeneration.     

Reprint of "on the size of the active site in proteases. I. Papain"

α-Synuclein is the major component of Lewy bodies and Lewy neurites, the pathological hallmarks of surviving neuronal cells in Parkinson's disease patients. However, the physiological role played by α-synuclein remains unclear. In this study, spectrin beta non-erythrocyte 1 (SPTBN1) interacted with α-synuclein in phage display assays using a normalized human brain cDNA library. A direct interaction between α-synuclein and SPTBN1 was confirmed by GST pull-down and co-immunoprecipitation assays. SPTBN1 and α-synuclein proteins colocalized in N2a neuronal cells. Transfection of SPTBN1 caused human SH-SY5Y dopaminergic neuron cells to inappropriately induce neurites, which extended from cell bodies. Cotransfection with α-synuclein reversed SPTBN1-induced excessive neurite branching in SH-SY5Y cells, and only a single neurite extended from each neuron. These results suggest that α-synuclein modulates neurite outgrowth by interacting with cytoskeletal proteins such as SPTBN1.     

Studies of Schwann cell proliferation. III. Evidence for the surface localization of the neurite mitogen

In the preceding paper (Salzer et al., 1980, J. Cell Biol. 84:753-- 766), evidence was presented that a neurite membrane fraction could be used to stimulate Schwann cell proliferation in culture. In this study, we present evidence that the mitogenic signal by which intact neurites or neurite membranes stimulate Schwann cell proliferation is located at the neurite surface. This conclusion is based on the following observations: (a) stimulation of Schwann cell proliferation by neurons requires direct contact between neurites and Schwann cells, separation of the two cells by a permeable collagen diaphragm 6 microns thick prevents Schwann cell proliferation; (b) treatment of intact neurites with trypsin before preparation of neurite membranes abolishes the ability of these membranes to be mitogenic for Schwann cells; and (c) the mitogenic activity of neurite homogenates is exclusively localized in the particulate rather than the soluble fraction of the homogenate. The mitogenic component on the neurite surface is heat labile, and is inactivated by aldehyde fixation. Preliminary data suggest that the mitogenic effect of neurite on Schwann cells is not mediated by 3',5'- cyclic AMP.     

Mechanical tension can specify axonal fate in hippocampal neurons

Here we asked whether applied mechanical tension would stimulate undifferentiated minor processes of cultured hippocampal neurons to become axons and whether tension could induce a second axon in an already polarized neuron. Experimental tension applied to minor processes produced extensions that demonstrated axonal character, regardless of the presence of an existing axon. Towed neurites showed a high rate of spontaneous growth cone advance and could continue to grow out for 1-3 d after towing. The developmental course of experimental neurites was found to be similar to that of unmanipulated spontaneous axons. Furthermore, the experimentally elongated neurites showed compartmentation of the axonal markers dephospho-tau and L-1 in towed outgrowth after 24 h. Extension of a second axon from an already polarized neuron does not lead to the loss of the spontaneous axon either immediately or after longer term growth. In addition, we were able to initiate neurites de novo that subsequently acquired axonal character even though spontaneous growth cone advance began while the towed neurite was still no longer than its sibling processes. This suggests that tension rather than the achievement of a critical neurite length determined axonal specification.     

Localization of SSeCKS in unmyelinated primary sensory neurons

Background

RhoA and Rho kinase inhibitors overcome the inhibition of axonal regeneration posed by central nervous system (CNS) substrates.

Methods

To investigate if inhibition of the Rho pathway augments the neurite extension that naturally occurs in the peripheral nervous system (PNS) following nerve damage, dorsal root ganglion neurons and Schwann cell co-cultures were incubated with culture medium, C3 fusion toxin, and the Rho kinase (ROCK) inhibitors Y27632 and H1152. The longest neurite per neuron were measured and compared. Incubation with Y27632 and H1152 resulted in significantly longer neurites than controls when the neurons were in contact with Schwann cells. When separated by a porous P.E.T. membrane, only the group incubated with H1152 developed significantly longer neurites. This work demonstrates that Rho kinase inhibition augments neurite elongation in the presence of contact with a PNS-like substrate.     

Dscam1-mediated self-avoidance counters netrin-dependent targeting of dendrites in Drosophila

Dendrites and axons show precise targeting and spacing patterns for proper reception and transmission of information in the nervous system. Self-avoidance promotes complete territory coverage and nonoverlapping spacing between processes from the same cell [1, 2]. Neurons that lack Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) show aberrant overlap, fasciculation, and accumulation of dendrites and axons, demonstrating a role in self-recognition and repulsion leading to self-avoidance [3-11]. Fasciculation and accumulation of processes suggested that Dscam1 might promote process spacing by counterbalancing developmental signals that otherwise promote self-association [9, 12]. Here we show that Dscam1 functions to counter Drosophila sensory neuron dendritic targeting signals provided by secreted Netrin-B and Frazzled, a netrin receptor. Loss of Dscam1 function resulted in aberrant dendrite accumulation at a Netrin-B-expressing target, whereas concomitant loss of Frazzled prevented accumulation and caused severe deficits in dendritic territory coverage. Netrin misexpression was sufficient to induce ectopic dendritic targeting in a Frazzled-dependent manner, whereas Dscam1 was required to prevent ectopic accumulation, consistent with separable roles for these receptors. Our results suggest that Dscam1-mediated self-avoidance counters extrinsic signals that are required for normal dendritic patterning, but whose action would otherwise favor neurite accumulation. Counterbalancing roles for Dscam1 may be deployed in diverse contexts during neural circuit formation.     

Motile areas of leech neurites are rich in microfilaments and two actin-binding proteins: gelsolin and profilin.

Cell motility is produced by changes in the dynamics and organization of actin filaments. The aim of the experiments described here was to test whether growing neurites contain two actin-binding proteins, gelsolin and profilin, that regulate polymerization of actin and affect non-neuronal cell motility. The distribution of gelsolin, profilin and the microfilaments was compared by immunocytochemistry of leech neurons growing in culture. We observed that microfilaments are enriched in the peripheral motile areas of the neurites. Both gelsolin and profilin are also concentrated in these regions. Gelsolin is abundant in filopodia and is associated with single identifiable microfilament bundles in lamellipodia. Profilin is not prominent in filopodia and shows a diffuse staining pattern in lamellipodia. The colocalization of gelsolin and profilin in motile, microfilament-rich areas supports the hypothesis that they synergistically regulate the actin dynamics that underlie neurite growth.     

Effect of the weaver (wv) mutation on cerebellar neuron differentiation. II. Quantitation of neuron behavior in culture

Quantitative measurements of neuron behavior from time-lapse microcinematography of dissociated cultures of normal (+/+), heterozygous weaver (+/wv), and homozygous weaver (wv/wv) 7-day-old mouse cerebellum were performed to identify dose-dependent expressions of the mutant allele. Impaired neurite growth by granule cell neurons is a direct result of a dose-dependent increased frequency of neurite retraction and decreased rate of growth cone advancement. The number of retractions per neurite is 0.2, 1.0, and 2.0 for +/+, +/wv, and wv/wv neurites, respectively. Maximal rates of growth cone advancement are 1041, 443, and 250 micron/day for +/+, +/wv, and wv/wv granule cell neurites, respectively. Neurite initiation is actually increased in wv/wv cultures, though the neurites are not well sustained. The frequency of neurite initiation is 1.0, 1.7, and 2.2 for +/+, +/wv, and wv/wv neurons, respectively. Measurements of oscillations of somal position revealed that the cell center moves increasing distances over short times in proportion to the number of mutant genes. Nuclear translocation, the mode of somal migration in vivo and in vitro, occurs at the same frequency and rate in normal and mutant cultures. Weaver gene expression induces a cytopathology affecting various morphogenetic events rather than producing a block at a specific stage in granule cell differentiation. It is hypothesized that the dose-dependent impairments of cell motility reflect weaver gene action at the cell surface or cytoskeleton.     

Direct interactions between immunocytes and neurons after axotomy in Aplysia

Axon growth during development and after injury has processes in common, but also differs in that regeneration requires the participation of cells of the immune system. To investigate how neuron-immunocyte interactions might influence regeneration, we developed an in vitro model whereby neurons and hemocytes from Aplysia californica were cocultured. The hemocytes, which behave like vertebrate macrophages, migrated randomly throughout the dish. When a neuron was encountered, some hemocytes exhibited an avoidance response, whereas others formed stable contacts. Hemocytes did not distinguish between neurons from different animals. Stable contacts occurred on neurites and growth cones, but not the cell soma, and were benign in that the hemocytes did not impede neurite growth. When hemocytes attached to the cell body, it presaged the destruction of the neuron. Destruction was a dynamic process that was initiated when groups of one to three hemocytes adhered to various regions of the cell soma. Each group was then joined by other hemocytes. They did not contact the neuron, but interconnected the initial groups, forming a network around the neuron. The network then contracted to dismember the cell. Once a neuron was destroyed, hemocytes removed the debris by phagocytosis. Both damaged neurons and those without apparent damage were targets for destruction. Severing neurites with a needle resulted in the destruction of only one of six cells. Our studies suggest that hemocytes, and by extrapolation, vertebrate macrophages, exhibit highly complex interactions with neurons that can exert a variety of influences on the course of nerve regeneration.