Neuronal contact guidance in magnetically aligned fibrin gels: effect of variation in gel mechano-structural properties.

Neurite outgrowth from chick dorsal root ganglia entrapped in isotropic and magnetically aligned fibrin gels was studied, and the dependence on the diameter of the fibrin fibrils was characterized. The fibrin fibril diameter was varied, as inferred from turbidity measurements, by using different Ca2+ concentrations in the fibrin-forming solution, but this variation was accomplished without affecting the degree of magnetic-induced alignment, as directly visualized in fluorescently spiked gels. Magnetically aligned fibrin gels possessing different fibril diameters but similar alignment resulted in drastic changes in the contact guidance response of neurites, with no response in gels formed in 1.2 mM Ca2+ (having smaller fibril diameter, ca. 150 nm), but a strong response in gels formed in 12 and 30 mM Ca2+ (having larger fibril diameter, ca. 510 nm) with an attendant two-fold increase in neurite length. These changes are attributed to variation of the mechano-structural properties of the network of aligned fibrils as the fibril diameter is varied.

Neurotrophins and the dynamic regulation of the neuronal cytoskeleton.

The morphology of neuronal axons and dendrites is dependent on the dynamics of the cytoskeleton. An understanding of neurodevelopment and adult neuroplasticity must therefore include a detailed description of the intrinsic and extrinsic mechanisms that regulate the organization and dynamics of actin filaments and microtubules. In this paper we review recent advances in the understanding of the dynamic regulation of neuronal morphology by interactions among cytoskeletal components and the regulation of the cytoskeleton by neurotrophins.

Stabilization of growing retinal axons by the combined signaling of nitric oxide and brain-derived neurotrophic factor.

The pattern of axonal projections early in the development of the nervous system lacks the precision present in the adult. During a developmental process of refinement, mistargeted projections are eliminated while correct projections are retained. Previous studies suggest that during development nitric oxide (NO) is involved in the elimination of mistargeted retinal axons, whereas brain-derived neurotrophic factor (BDNF) may stabilize retinal axon arbors. It is unclear whether these neuromodulators interact. This study showed that NO induced growth cone collapse and retraction of developing retinal axons. This effect was not attributable to NO-induced neurotoxicity. BDNF protected growth cones and axons from the effects of NO. This effect was specific to BDNF, because neither nerve growth factor (NGF) nor neurotrophin-3 (NT-3) prevented NO-induced growth cone collapse and axon retraction. Exposure to both BDNF and NO, but not either factor alone, stabilized growth cones and axons. Stabilized axons exhibited minimal retraction or extension. This response appears to be a new axon "state" and not simply a partial amelioration of the effect of NO, because lower doses of BDNF or NO allowed axon extension. Furthermore, BDNF/NO-induced growth cone stabilization correlated with the appearance of a cytochalasin D-resistant population of actin filaments. BDNF protection from NO likely was mediated locally at the level of the growth cone, because growth cones or individual filopodia in contact with BDNF-coated beads were protected from NO-induced collapse. These findings suggest a cellular mechanism by which some axonal connections are stabilized and some are eliminated during development.

Embryonic neurons adapt to the inhibitory proteoglycan aggrecan by increasing integrin expression.

The primary mediators of cell migration during development, wound healing and metastasis, are receptors of the integrin family. In the developing and regenerating nervous system, chondroitin sulfate proteoglycans (CSPGs) inhibit the integrin-dependent migration of neuronal growth cones. Here we report that embryonic sensory neurons cultured on the growth-promoting molecule laminin in combination with the inhibitory CSPG aggrecan rapidly adapt to inhibition. Adaptation is associated with a two- to threefold increase in the levels of RNA and surface protein for two laminin receptors, integrin alpha6beta1 and alpha3beta1, indicating that integrin expression is regulated by aggrecan. Increased integrin expression is associated both with increases in neuronal cell adhesion/outgrowth and with decreases in the ability of aggrecan to inhibit cell adhesion. Directly increasing integrin expression by adenoviral infection is sufficient to eliminate the inhibitory effects of aggrecan, indicating that upregulation of integrin receptors may promote neuronal regeneration in the presence of inhibitory matrix components.

Guided neurite elongation and schwann cell invasion into magnetically aligned collagen in simulated peripheral nerve regeneration.

High-strength magnetic fields were used to align collagen gel formed into 4-mm-diameter rods during the self-assembly of type I collagen monomers into fibrils. We developed an in vitro assay to study neurite elongation into the magnetically aligned collagen gel rods from dorsal root ganglia (DRG) explants placed onto one end of the rods. The depth of neurite elongation from chick embryo DRG neurons into these rods was found to be substantially greater than that observed in controls and increased with an increase in magnetic field strength, as did the collagen gel rod birefringence, indicative of collagen fibril alignment along the rod axis. Moreover, the axial bias of neurite elongation became more pronounced with an increase in magnetic field strength, presumably due to a contact guidance response of growth cones at the neurite tips. Coinvasion of Schwann cells from neonatal rat DRG was also studied in these assays using double immunolabeling. In the absence of serum, Schwann cells were highly associated with, and often trailed, elongating neurites. In the presence of serum, Schwann cells showed significantly higher rates of invasion and formed axially aligned chords reminiscent of bands of Büngner. These results may translate into an improved method of entubulation repair of transected peripheral nerves by directing and stimulating axonal growth through a tube filled with magnetically aligned collagen gel.

Axon guidance: A balance of signals sets axons on the right track.

Axon guidance depends on the transduction of extracellular guidance cues into motile responses by the axonal growth cone. Recent studies in vivo have elucidated mechanisms required for this process that involve kinases and phosphatases, calcium dynamics and remodeling of the actin cytoskeleton.

Different contributions of microtubule dynamics and transport to the growth of axons and collateral sprouts.

Axonal growth is believed to depend on microtubule transport and microtubule dynamic instability. We now report that the growth of axon collateral branches can occur independent of microtubule dynamic instability and can rely mostly on the transport of preassembled polymer. Raising embryonic sensory neurons in concentrations of either taxol or nocodazole (NOC) that largely inhibit microtubule dynamics significantly inhibited growth of main axonal shafts but had only minor effects on collateral branch growth. The collaterals of axons raised in taxol or nocodazole often contained single microtubules with both ends clearly visible within the collateral branch ("floating" microtubules), which we interpret as microtubules undergoing transport. Furthermore, in these collaterals there was a distoproximal gradient in microtubule mass, indicating the distal accumulation of transported polymer. Treatment of cultures with a high dose of nocodazole to deplete microtubules from collaterals, followed by treatment with 4-20 nM vinblastine to inhibit microtubule repolymerization, resulted in the time-dependent reappearance and subsequent distal accumulation of floating microtubules in collaterals, providing further evidence for microtubule transport into collateral branches. Our data show that, surprisingly, the contribution of microtubule dynamics to collateral branch growth is minor compared with the important role of microtubule dynamics in growth cone migration, and they indicate that the transport of microtubules may provide sufficient cytoskeletal material for the initial growth of collateral branches.

Localized sources of neurotrophins initiate axon collateral sprouting.

The sprouting of axon collateral branches is important in the establishment and refinement of neuronal connections during both development and regeneration. Collateral branches are initiated by the appearance of localized filopodial activity along quiescent axonal shafts. We report here that sensory neuron axonal shafts rapidly sprout filopodia at sites of contact with nerve growth factor-coated polystyrene beads. Some sprouts can extend up to at least 60 micro(m) through multiple bead contacts. Axonal filopodial sprouts often contained microtubules and exhibited a debundling of axonal microtubules at the site of bead-axon contact. Cytochalasin treatment abolished the filopodial sprouting, but not the accumulation of actin filaments at sites of bead-axon contact. The axonal sprouting response is mediated by the trkA receptor and likely acts through a phosphoinositide-3 kinase-dependent pathway, in a manner independent of intracellular Ca2+ fluctuations. These findings implicate neurotrophins as local cues that directly stimulate the formation of collateral axon branches.

Axon guidance: GTPases help axons reach their targets.

During development and regeneration of the nervous system, axons must correctly navigate to their specific targets through a complex molecular environment. Recent work has shed light on how GTPases of the Ras family are involved in transducing extracellular signals into responses that lead to directed neurite outgrowth.

Micropatterning gradients and controlling surface densities of photoactivatable biomolecules on self-assembled monolayers of oligo(ethylene glycol) alkanethiolates.

BACKGROUND: Bioactive molecules that are covalently immobilized in patterns on surfaces have previously been used to control or study cell behavior such as adhesion, spreading, movement or differentiation. Photoimmobilization techniques can be used, however, to control not only the spatial pattern of molecular immobilization, termed the micropattern, but also the surface density of the molecules--a characteristic that has not been previously exploited. RESULTS: Oligopeptides containing the bioactive Arg-Gly-Asp cell-adhesion sequence were immobilized upon self-assembled monolayers of an oligo(ethylene glycol) alkanethiolate in patterns that were visualized and quantified by autoradiography. The amount and pattern of immobilized peptide were controlled by manipulating the exposure of the sample to a UV lamp or a laser beam. Patterns of peptides, including a density gradient, were used to control the location and number of adherent cells and also the cell shape. CONCLUSIONS: A photoimmobilization technique for decorating surfaces with micropatterns that consist of variable densities of bioactive molecules is described. The efficacy of the patterns for controlling cell adhesion and shape has been demonstrated. This technique is useful for the study of cell behavior on micropatterns.

Concentration-dependent stimulation and inhibition of growth cone behavior and neurite elongation by protein kinase inhibitors KT5926 and K-252a.

We examined the concentration- and time-dependent effects of two related protein kinase inhibitors, KT5926 and K-252a, on neurite formation and nerve growth cone migration of chick embryo sensory neurons. The effects of these drugs on neurite formation over an 18-h period were dissimilar. KT5926 stimulated neurite formation at concentrations between 100 and 500 nM and inhibited neurite formation at 5 microM. K-252a had no stimulatory effects on neurite formation, and it inhibited neurite formation at concentrations above 50 nM. This difference may occur because K-252a inhibits activation of the nerve growth factor receptor trk A, while KT5926 does not inhibit trk A. Both drugs, however, had similar immediate effects on growth cone migration. Growth cone migration and lamellipodial spreading were rapidly stimulated by 500 nM concentrations of KT5926 and K-252a. At 2 microM levels of either drug, growth cone spreading was still stimulated, but growth cone migration was inhibited by both drugs. These results show that changes in protein phosphorylation/dephosphorylation can rapidly regulate the cellular machinery that is responsible for driving growth cone migration and neurite elongation. The different effects of 2 microM concentrations of either KT5926 or K-252a on growth cone spreading versus migration suggests that the actin-dependent protrusive motility of the growth cone leading margin is regulated differently by changes in protein phosphorylation and dephosphorylation than the cytoskeletal mechanism that drives neurite elongation.

The trkA receptor mediates growth cone turning toward a localized source of nerve growth factor.

We have developed an in vitro system for studying the interaction of chick dorsal root ganglion neuronal growth cones with a localized source of nerve growth factor (NGF) covalently conjugated to polystyrene beads. Growth cones rapidly turned and migrated under NGF-coated beads in a process that involved the initial formation of persistent contact with a bead, followed by directed flow of cytoplasm toward the point of contact. A role for the local activation of the high-affinity NGF receptor trkA was suggested by a strong inhibition of the turning response by (1) the addition of an antibody against the extracellular portion of trkA, (2) the elevation of the background concentration of NGF to saturate trkA, or (3) the presence of a concentration of the drug K252a that inhibits trkA activation. NGF binding to the pan-neurotrophin receptor p75 is also involved but is not required for turning. These data show a new role for both the trkA and the p75 receptors: the mediation of local events in the guidance of nerve growth cones.

Dynamic microtubule ends are required for growth cone turning to avoid an inhibitory guidance cue.

Growth cone turning is an important mechanism for changing the direction of neurite elongation during development of the nervous system. Our previous study indicated that actin filament bundles at the leading margin direct the distal microtubular cytoskeleton as growth cones turn to avoid substratum-bound chondroitin sulfate proteoglycan. Here, we investigated the role of microtubule dynamics in growth cone turning by using low doses of vinblastine and taxol, treatments that reduce dynamic growth and shrinkage of microtubule ends. We used time-lapse phase-contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones as they encountered a border between fibronectin and chondroitin sulfate proteoglycan in the presence and absence of 4 nM vinblastine or 7 nM taxol. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and microtubules containing tyrosinated and detyrosinated alpha-tubulin. Our results show that after contact with substratum-bound chondroitin sulfate proteoglycan, vinblastine- and taxol-treated growth cones did not turn, as did controls; instead, they stopped or sidestepped. Even before drug-treated growth cones contacted a chondroitin sulfate proteoglycan border, they were narrower than controls, and the distal tyrosinated microtubules were less splayed and were closer to the leading edges of the growth cones. We conclude that the splayed dynamic distal ends of microtubules play a key role in the actin filament-mediated steering of growth cone microtubules to produce growth cone turning.

Ligand-induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth.

Receptors of the integrin family are expressed by every cell type and are the primary means by which cells interact with the extracellular matrix. The control of integrin expression affects a wide range of developmental and cellular processes, including the regulation of gene expression, cell adhesion, cell morphogenesis and cell migration. Here we show that the concentration of substratum-bound ligand (laminin) post-translationally regulates the amount of receptor (alpha6beta1, integrin) expressed on the surface of sensory neurons. When ligand availability is low, surface amounts of receptor increase, whereas integrin RNA and total integrin protein decrease. Ligand concentration determines surface levels of integrin by altering the rate at which receptor is removed from the cell surface. Furthermore, increased expression of integrin at the cell surface is associated with increased neuronal cell adhesion and neurite outgrowth. These results indicate that integrin regulation maintains neuronal growth-cone motility over a broad range of ligand concentrations, allowing axons to invade different tissues during development and regeneration.

Actin filament bundles are required for microtubule reorientation during growth cone turning to avoid an inhibitory guidance cue.

The extracellular matrix through which growth cones navigate contains molecules, such as chondroitin sulfate proteoglycan, that can inhibit growth cone advance and induce branching and turning. Growth cone turning is accompanied by rearrangement of the cytoskeleton. To identify changes in the organization of actin filaments and microtubules that occur as growth cones turn, we used time-lapse phase contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones at a substratum border between fibronectin and chondroitin sulfate proteoglycan, in the presence and absence of cytochalasin B. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and dynamic and stable microtubules. Our results suggest that microtubules are rearranged within growth cones to accomplish turning to avoid chondroitin sulfate proteoglycan. Compared to growth cones migrating on fibronectin, turning growth cones were more narrow, and they contained dynamic microtubules that were closer to the leading edge and were more bundled. Cytochalasin B-treated growth cones sidestepped laterally along the border instead of turning, and in sidestepping growth cones, microtubules were not bundled and aligned. We conclude that actin filament bundles are required for microtubule reorientation and growth cone turning to avoid chondroitin sulfate proteoglycan.

Growth cone behavior in the presence of soluble chondroitin sulfate proteoglycan (CSPG), compared to behavior on CSPG bound to laminin or fibronectin.

Proteoglycans (PGs) are complex macromolecules of the extracellular matrix (ECM) that have a wide variety of effects on developing and regenerating neurons in vivo and in vitro. One hypothesis regarding the mechanisms of PG regulation of neuronal behavior states that the conformation of PGs may be critical, and thus that ECM- or cell surface-bound PGs may operate differently than secreted (soluble) PGs. Therefore, this study examined differences between the effects of soluble chondroitin sulfate proteoglycan (CSPG) and substratum-bound CSPG on neuronal growth cone behavior. Dissociated chicken dorsal root ganglion (DRG) neurons were cultured on either laminin (LN) or fibronectin (FN), both sensory neurite outgrowth-promotin glycoproteins. CSPG (or chondroitin sulfate alone) was either bound to FN or LN, or was added to the culture media. Subsequently, using time lapse video microscopy and image analysis, this study measured: (1) neuronal attachment, (2) neurite outgrowth, (3) rate of neurite elongation, and (4) filopodial length and lifespan. To determine the site of CSPG action, DRG neurons were grown on either: CS-1, a FN peptide [Humphries M. J. et al. (1987) J. biol. Chem. 262, 6886-6892], or a recombinant FN protein, RFNIIIcs (Maejne, submitted), both of which permit DRG attachment and outgrowth but do not have recognized CSPG binding sites, and the resulting neuronal behavior was compared to that of DRG neurons grown on intact FN. The results of these studies confirm that the effect of CSPG on DRG neurons is concentration-, conformation- and substratum-dependent. On I.N, soluble CSPG had little to no effect on neurite initiation or outgrowth, while substratum-bound CSPG inhibited neurite outgrowth. In contrast, on FN, soluble CSPG inhibited neurite outgrowth and decreased the rate of neurite elongation. Soluble CSPG did not affect the length of sensory growth cone filopodia or filopodial lifespan on either LN or FN. From the FN fragment experiments, we found that: (1) soluble CSPG reduces neurite outgrowth on FN or FN fragments, but not on LN, up to 80%, and reduces elongation rate on FN up to 50%, and (2) soluble CSPG regulates neuronal behavior by binding directly to growth cones elongating on FN. Given that substratum-bound CSPG from a variety of sources is inhibitory to neurite outgrowth and to the rate of neurite elongation, while soluble CSPG often has different effects on growth cone behavior, the regulation of growth cone behavior by CSPGs may be dependent upon CSPG conformation. Further, CSPG may affect growth cone behavior by either binding to the substratum or by binding directly to growth cones.

Expression of alpha 6 and beta 4 integrins in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin.

The surface of a normal ovary is covered by a monolayer of epithelial cells that rest on a basement membrane. The glycoprotein laminin is the major noncollagenous protein present in the basement membrane. The integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4 serve as cell surface receptors for laminin. During the progression of serous ovarian carcinoma, tumor cells are frequently exfoliated from the surface of the ovary, thereby losing contact with the basement membrane. This study was designed to determine whether alterations in integrin expression may be associated with the malignant phenotype of the primary ovarian tumor and exfoliated ovarian carcinoma cells in the ascites fluid. By immunohistochemical staining, the entire surface of epithelial cells of normal ovaries stained positively for beta 1, alpha 2, and alpha 3 integrins, whereas only the basal surface of the epithelial cells, where they are in contact with laminin, stained positively for alpha 6 and beta 4. The entire surface of epithelial cells of solid tumors from patients with serous ovarian carcinoma stained positively for beta 1, alpha 2, and alpha 3 integrins. In most cases, no intact basement membrane surrounded the tumor nests, and staining for alpha 6 and beta 4 was irregular. When present, the basement membrane stained positively for laminin, and the basal surface of the epithelial cells stained positively for alpha 6 and beta 4. Ovarian carcinoma ascites cells exhibited a distinct phenotype, with a significant decrease in expression of the alpha 6 and beta 4 integrin subunits. As alpha 6 and beta 4 integrin subunits are present at the basal surface of many epithelial cells and serve as receptors for laminin, it is possible that ovarian carcinoma epithelial cells may be released from the basement membrane of the ovary due to their deficit of alpha 6 and beta 4 integrin subunits.

Chick sensory neuronal growth cones distinguish fibronectin from laminin by making substratum contacts that resemble focal contacts.

The adhesive interactions of nerve growth cones stabilize elongating nerve fibers and mediate transmembrane signaling to regulate growth cone behaviors. We used interference reflection microscopy and immunocytochemistry to examine the dynamics and composition of substratum contacts that growth cones of chick sensory neurons make with extracellular adhesive glycoproteins, fibronectin and laminin. Interference reflection microscopy indicated that sensory neuronal growth cones on fibronectin-treated substrata, but not on laminin, make contacts that have the appearance and immobility of fibroblastic focal contacts. Interference reflection microscopy and subsequent immunocytochemical staining showed that beta 1 integrin and phosphotyrosine residues were concentrated at growth cone sites that resemble focal contacts. Two other components of focal contacts, paxillin and zyxin, were also co-localized with concentrated phosphotyrosine residues at sites that resemble focal contacts. Such staining patterns were not observed on laminin-treated substrata. Growth cone migration on fibronectin-treated substrata was inhibited by herbimycin A, a tyrosine kinase inhibitor. We conclude that sensory neuronal growth cones distinguish fibronectin from laminin by making contacts with distinct organization and regulation of cytoskeletal components at the adhesive sites. This finding suggests that growth cone interactions with different adhesive molecules lead to distinctive transmembrane organization and signaling to regulate nerve fiber elongation.

The cytoskeleton in nerve growth cone motility and axonal pathfinding.

Axonal pathfinding occurs through detection of environmental cues by cytoskeletal machinery that is responsible for growth cone migration. The cycle of filopodial and lamellipodial protrusion, adhesion, and generation of tensions to advance a growth cone result from concerted actions of ABPs to regulate actin filament polymerization, assembly into networks and bundles, and production of tension to move the growth cone and its contents. The direction of neurite elongation is controlled by forward movement of microtubules in growth cones, which is pioneered by the advance of microtubules into P domain of the leading margin. Actin filaments both promote and impede this advance of microtubules in several ways. This cytoskeletal machinery is controlled by major signaling mechanisms. To understand growth cone guidance we must reveal the spatial and temporal changes generated in [Ca++]i, phospholipids, and protein phosphorylation and dephosphorylation, and then identify the ABPs and MAPs that are their targets.

Characterization of spontaneous calcium transients in nerve growth cones and their effect on growth cone migration.

This study examines the mechanisms of spontaneous and induced [Ca2+]i spiking in nerve growth cones and the effect of spikes on growth cone migration. Over a 10-20 min observation period, 29% of DRG growth cones undergo spontaneous and transient elevations in physiological extracellular Ca2+ ((Ca2+)o; 2 mM), whereas 67% of growth cones exposed to 20 mM (Ca2+)o exhibit similar [Ca2+]i spikes. Spontaneous [Ca2+]i spiking was not observed in neuronal cell bodies or nonneuronal cells. Ca2+ influx through non-voltage-gated Ca2+ channels was required for spontaneous [Ca2+]i spikes in growth cones, since removal of (Ca2+)o, or addition of the general Ca2+ channel blockers La3+ or Ni2+, reversibly blocked [Ca2+]i spiking, while blockers of the voltage-gated Ca2+ channels did not. Experiments using agents that influence intracellular Ca2+ stores suggest that Ca2+ stores may buffer and release Ca2+ during growth cone [Ca2+]i spikes. Growth cone migration was immediately and transiently inhibited by [Ca2+]i spikes, but eventually returned to prespike rates.