Inhibition Of Neurite Extension Research Articles

Protein kinase C mediates neurite guidance at an astrocyte boundary.

During development, astrocytes play an active role in directing axons to their final targets. This guidance has been attributed in part to the increased expression of guidance molecules, such as tenascin-C and chondroitin sulfate proteoglycans, by boundary-forming astrocytes. We have previously used a culture model of astrocyte boundaries to demonstrate that neurites growing on permissive astrocytes alter their trajectory as they encounter less-permissive astrocytes. The present study investigated the role of the protein kinase C (PKC) family of signal transduction molecules in this form of axonal guidance. Neurons were plated onto mixed astrocyte monolayers in the presence of agents that either downregulate the phorbol ester-sensitive PKC isoforms or inhibit PKC. Both downregulation and inhibition of PKC increased the percentage of neurons that crossed onto the nonpermissive astrocytes. On astrocyte monolayers, phorbol ester modulation of PKC but not PKC inhibitors resulted in a decrease in overall neurite extension. PKC inhibitors also caused a similar alteration in the neuronal response to cell-free boundaries, at concentrations that did not inhibit neurite extension. Thus, phorbol-ester-sensitive PKC isoforms direct the guidance of neurites by astrocyte-derived matrix molecules.

COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

The developmental expression patterns of the nuclear orphan receptors COUP-TFs (chicken ovalbumin upstream promoter-transcription factors) have been correlated to neurogenesis in several animal species. Nevertheless, the role of COUP-TFs in neurogenesis remains unknown. We have studied the functional involvement of COUP-TFI in retinoic acid (RA)-induced neuronal differentiation of P19 embryonal carcinoma cells through two complementary approaches: 1) deregulated expression of COUP-TFI, and 2) inactivation of endogenous COUP-TFs by means of a dominant-negative COUP-TFI mutant. Low levels of wild-type (wt)COUP-TFI transgene expression did not inhibit neural cell fate and primarily enhanced neuron outgrowth from RA-treated P19 aggregates. In contrast, high COUP-TFI expression impeded the neuronal differentiation of P19 cells induced with RA, resulting in cell cultures lacking neurons. This morphological effect was correlated to an elevated level of E-cadherin mRNA. The dominant-negative COUP-TFI mutant induced cell packing after RA treatment and inhibited neurite extension and neuron outgrowth from aggregates. A RGD peptide interference assay indicated that endogenous COUP-TFs could favor migration of neurons through an integrin-dependent mechanism. Accordingly, vitronectin mRNA levels were shown to be up-regulated by COUP-TFI by RT-PCR analysis, and COUP-TFI stimulated the mouse vitronectin promoter activity in transient transfection assays. Taken together, these data indicate that COUP-TFI is not simply a global repressor of retinoid functions, but shows a high selectivity for regulating genes involved in cellular adhesion and migration processes that are particularly important for neuronal differentiation.

Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building.

Kinesin superfamily proteins (KIFs) comprise several dozen molecular motor proteins. The KIF3 heterotrimer complex is one of the most abundantly and ubiquitously expressed KIFs in mammalian cells. To unveil the functions of KIF3, microinjection of function-blocking monovalent antibodies against KIF3 into cultured superior cervical ganglion (SCG) neurons was carried out. They significantly blocked fast axonal transport and brought about inhibition of neurite extension. A yeast two-hybrid binding assay revealed the association of fodrin with the KIF3 motor through KAP3. This was further confirmed by using vesicles collected from large bundles of axons (cauda equina), from which membranous vesicles could be prepared in pure preparations. Both immunoprecipitation and immunoelectron microscopy indicated the colocalization of fodrin and KIF3 on the same vesicles, the results reinforcing the evidence that the cargo of the KIF3 motor consists of fodrin-associating vesicles. In addition, pulse-labeling study implied partial comigration of both molecules as fast flow components. Taken together, the KIF3 motor is engaged in fast axonal transport that conveys membranous components important for neurite extension.

Regulation of Calcineurin by Growth Cone Calcium Waves Controls Neurite Extension

Growth cones generate spontaneous transient elevations of intracellular Ca(2+) that regulate the rate of neurite outgrowth. Here we report that these Ca(2+) waves inhibit neurite extension via the Ca(2+)-dependent phosphatase calcineurin (CN) in Xenopus spinal neurons. Pharmacological blockers of CN (cyclosporin A and deltamethrin) and peptide inhibitors of CN [the Xenopus CN (xCN) autoinhibitory domain and African swine fever virus protein A238L] block the Ca(2+)-dependent reduction of neurite outgrowth in cultured neurons. Time-lapse microscopy of growing neurites demonstrates directly that the reduction in the rate of outgrowth by Ca(2+) transients is blocked by cyclosporin A. In contrast, expression of a constitutively active form of xCN in the absence of waves results in shorter neurite lengths similar to those seen in the presence of waves. The developmental expression pattern of xCN transcripts in vivo coincides temporally with axonal pathfinding by spinal neurons, supporting a role of CN in regulating Ca(2+)-dependent neurite extension in the spinal cord. Ca(2+) wave frequency and Ca(2+)-dependent expression of GABA are not affected by inhibition or activation of CN. However, phosphorylation of the cytoskeletal element GAP-43, which promotes actin polymerization, is reduced by Ca(2+) waves and enhanced by suppression of CN activity. CN ultimately acts on the growth cone actin cytoskeleton, because disrupting actin microfilaments with cytochalasin D or stabilizing them with jasplakinolide negates the effects of suppressing or activating CN. Destabilization or stabilization of microtubules with colcemide or taxol results in Ca(2+)-independent inhibition of neurite outgrowth. The results identify components of the cascade by which Ca(2+) waves act to regulate neurite extension.

The Alzheimer-related gene presenilin 1 facilitates notch 1 in primary mammalian neurons

The normal functional neurobiology of the Alzheimer's disease (AD) related gene presenilin 1 (PS1) is unknown. One clue comes from a genetic screen of Caenorhabditis elegans, which reveals that the presenilin homologue sel-12 facilitates lin-12 function [D. Levitan, I. Greenwald, Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer's disease gene, Nature 377 (1995) 351–355]. The mammalian homologue of lin-12, Notch1, is a transmembrane receptor that plays an important role in cell fate decisions during development, including neurogenesis, but does not have a known function in fully differentiated cells. To better understand the potential role of Notch1 in mammalian postmitotic neurons and to test the hypothesis that Notch and PS 1 interact, we studied the effect of Notch1 transfection on neurite outgrowth in primary cultures of hippocampal/cortical neurons. We demonstrate that Notch1 inhibits neurite extension, and thus has a function in postmitotic mature neurons in the mammalian CNS. Furthermore, we present evidence demonstrating that there is a functional interaction between PS1 and Notch1 in mammalian neurons, analogous to the sel-12/lin-12 interaction in vulval development in C. elegans [D. Levitan, T. Doyle, D. Brousseau, M. Lee, G. Thinakaran, H. Slunt, S. Sisodia, I. Greenwald, Assessment of normal and mutant human presenilin function in Caenorhabditis elegans, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 14940–14944; D. Levitan, I. Greenwald, Effect of Sel-12 presenilin on Lin-12 localization and function in Caenorhabditis elegans, Development, 125 (1998) 3599–3606]. The inhibitory effect of Notch1 on neurite outgrowth is markedly attenuated in neurons from PS1 knockout mice, and enhanced in neurons from transgenic mice overexpressing wild type PS1, but not mutant PS1. These data suggest that PS1 facilitates Notch1 function in mammalian neurons, and support the hypothesis that a functional interaction exists between PS1 and Notch1 in postmitotic mammalian neurons.

Distribution of synaptosomal-associated protein 25 in nerve growth cones and reduction of neurite outgrowth by botulinum neurotoxin A without altering growth cone morphology in dorsal root ganglion neurons and PC-12 cells

Synaptosomal-associated protein 25 has been regarded as one of the target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptors essential for exocytosis of vesicles in synapses. We have previously reported that cleavage of syntaxin, which is another target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, with botulinum neurotoxin C1 resulted in inhibition of neurite extension and morphological changes including growth cone collapse and large vacuole formation. As an attempt to explore the mechanism of growth cone extension, we examined the ultrastructural localization of synaptosomal-associated protein 25 in growth cones with or without treatment of botulinum neurotoxin A, which cleaves synaptosomal-associated protein 25. In dorsal root ganglion neurons, light microscopy demonstrated synaptosomal-associated protein 25 immunoreactivity throughout the neurons, including the cell bodies, neurites and growth cones. Using electron microscopy, gold signals immunoreactive for synaptosomal-associated protein 25 were identified diffusely in the cytoplasm of the growth cones. In contrast, in PC-12 cells, a large number of gold signals were localized on the plasma membranes. High levels of signal were also found in the cytoplasm in the central region of the growth cones. We also confirmed that botulinum neurotoxin A treatment reduced neurite extension by about 50%. However, both in dorsal root ganglion neurons and in PC-12 cells we found no differences in the ultrastructure nor in the localization of synaptosomal-associated protein 25 between growth cones with and without toxin treatment. These results indicate that cleavage of synaptosomal-associated protein 25 inhibits growth cone extension in a manner different than that of syntaxin cleavage. The results of this study suggest the possibility that synaptosomal-associated protein 25 is involved in growth cone extension through a process independent of vesicle fusion.

Regulation of nerve growth mediated by inositol 1,4,5-trisphosphate receptors in growth cones.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) acts as a Ca2+ release channel on internal Ca2+ stores. Type 1 IP3R (IP3R1) is enriched in growth cones of neurons in chick dorsal root ganglia. Depletion of internal Ca2+ stores and inhibition of IP3 signaling with drugs inhibited neurite extension. Microinjection of heparin, a competitive IP3R blocker, induced neurite retraction. Acute localized loss of function of IP3R1 in the growth cone induced by chromophore-assisted laser inactivation resulted in growth arrest and neurite retraction. IP3-induced Ca2+ release in growth cones appears to have a crucial role in control of nerve growth.

Cholinesterase Inhibitors Induce Growth Cone Collapse and Inhibit Neurite Extension in Primary Cultured Chick Neurons

Cholinesterase inhibitors are commonly prescribed medicines in neurological and anesthesiological clinical practices at relatively high doses. Recent studies report that cholinesterases, originally described as neurotransmitter degradation enzymes, are expressed in the developing central nervous systems. They are hypothesized to play an important role during the establishment of neuronal cytoarchitecture. In this study, the effects of several clinically utilized cholinesterase inhibitors on one potential aspect of neuronal development were examined using a primary culture system of chick central and peripheral neurons. Three cholinesterase inhibitors, physostigmine, neostigmine, and edrophonium, suppressed the activity of the leading edges of the extending axons (the nerve growth cones) dose dependently. Filopoda and lamellipodia of nerve growth cones were collapsed by the exposure to the cholinesterase inhibitors. This action was quick, mostly within 5 min, and partly irreversible. The lowest concentration that could induce statistically significant growth cone collapse was 10(-5) M for physostigmine, 10(-4) M for neostigmine, and 10(-2) for edrophonium, respectively. The ED50 values for the growth cone collapsing activity were approximately, 10(-3) M for physostigmine. 10(-2.5) M for neostigmine, and 10(-2) M for edrophonium both in dorsal root ganglion culture and in retinal culture. Even though the result of this in vitro study cannot be applied directly to the in vivo situation, physicians should consider the potential detrimental effects of cholinesterase inhibitors to growing and regenerating nervous tissues.

Comparison of Neurite Outgrowth Induced by Intact and Injured Sciatic Nerves: A Confocal and Functional Analysis

Mechanisms regulating axon growth in the peripheral nervous system have been studied by means of an in vitro bioassay, the tissue section culture, in which regenerating neurons are grown on substrata made up of tissue sections. Sections from intact and degenerated sciatic nerves proved to be different in their ability to support neurite outgrowth of embryonic chick sensory neurons from both qualitative and quantitative points of view. On denervated nerve sections, the total length of neurites elaborated per neuron was almost twice that found on intact nerve sections. In addition, confocal microscopy revealed a striking difference between intact and denervated nerve substrata: on denervated nerve sections, neurites grew inside the internal structures of endoneurial Schwann cell tubes, within the underlying tissue sections, whereas on intact nerve sections neurites extended along endoneurial basal laminae but never entered Schwann cell tubes. Perturbation experiments were used to analyze some of the molecular determinants that control neurite outgrowth in this system. Antibodies directed against the beta1-integrin subunit inhibited neurite extension on both normal and degenerated rat sciatic nerve tissue. Strikingly, however, differential inhibition was observed using antibodies directed against extracellular matrix molecules. Anti-laminin-2 (merosin) antibodies drastically reduced both the percentage of growing neurons and the total length of neurites on denervated nerve sections, but they did not modify these parameters on sections of normal nerve. Taken together, these results suggest that laminin-2/merosin promotes neurite outgrowth in peripheral nerve environments but only after Wallerian degeneration, which is when axons are allowed to extend within endoneurial tubes.

The influence of physical structure and charge on neurite extension in a 3D hydrogel scaffold

Understanding neural cell differentiation and neurite extension in three-dimensional scaffolds is critical for neural tissue engineering. This study explores the structure-function relationship between a 3D hydrogel scaffold and neural cell process extension and examines the role of ambient charge on neurite extension in 3D scaffolds. A range of agarose hydrogel concentrations was used to generate varied gel physical structures and the corresponding neurite extension was examined. Agarose gel concentration and the corresponding pore radius are important physical properties that influence neural cell function. The average pore radii of the gels were determined while the gel was in the hydrated state and in two different dehydrated states. As the gel concentration was increased, the average pore radius decreased exponentially. Similarly, the length of neurites extended by E9 chick DRGs cultured in agarose gels depends on gel concentration. The polycationic polysaccharide chitosan and the polyanionic polysaccharide alginate were used to incorporate charge into the 3D hydrogel scaffold, and neural cell response to charge was studied. Chitosan and alginate were covalently bound to the agarose hydrogel backbone using the bi-functional coupling agent 1,1'carbonyldiimldazole. DRGs cultured in chitosan-coupled agarose gel exhibited a significant increase in neurite length compared to the unmodified agarose control. Conversely, the alginate-coupled agarose gels significantly inhibited neurite extension. This study demonstrates a strong, correlation between the ability of sensory ganglia to extend neurites in 3D gels and the hydrogel pore radius. In addition, our results demonstrate that charged biopolymers influence neurite extension in a polarity dependent manner.

Transfection of neonatal rat cochlear cells in vitro with an adenovirus vector

A recombinant adenovirus vector containing a βI-galactosidase reporter gene was used to transfect neonatal rat organ of Corti or spiral ganglion explants in vitro. Infection at appropriate titers (106−107 pfu/ml) transduced virtually all cells in the cultures after 72 hr. However, spiral ganglion neurons and cells in the inner hair cell regions of the organ of Corti showed the highest levels of expression. Viral titers that produced high levels of β-galactosidase expression did not appear to damage the cultures, and did not inhibit neurite outgrowth from spiral ganglion cells. However, higher titers (108-109pfu/ml) clearly diminished explant viability and inhibited neurite extension. The results demonstrate that cochlear cells can be transfected successfully with an adenovirus vector, at viral titers which do not induce obvious signs of cellular damage or dysfunction.

BAPTA-AM and ethanol protect cerebellar granule neurons from the destructive effect of the weaver gene.

The mechanisms by which the weaver gene (Reeves et al., 1989; Patil et al., 1995) inhibits neurite extension and/or induces death of the granule neurons in homozygous weaver mouse cerebellum are not presently understood. Here we show that BAPTA-AM and ethanol, which either reduce cytosolic levels of free calcium or prevent calcium entry, promote neurite outgrowth of the weaver neurons similar to the L-type calcium channel blocker verapamil (Liesi and Wright, 1996). Importantly, BAPTA-AM, ethanol, and verapamil not only restore neurite outgrowth of the weaver neurons but adjust their depolarized resting membrane potentials to the levels of normal neurons. These results indicate that calcium-dependent mechanisms mediate the action of the weaver gene and that the weaver neurons can be normalized by blocking this calcium effect. We further report that BAPTA-AM and verapamil also have a neuroprotective effect on normal neurons exposed to high concentrations of ethanol. We suggest that verapamil should be evaluated as a drug for treatment of alcohol-induced brain damage and neurodegenerative disorders.

Inhibition of motor axon growth by T-cadherin substrata.

As spinal motor neurons project to their hindlimb targets, their growth cones avoid particular regions along their pathway. T-cadherin is discretely distributed in the avoided caudal sclerotome and on extrasynaptic muscle surfaces (B. J. Fredette and B. Ranscht (1994) J. Neurosci. 14, 7331-7346), and therefore, the ability of T-cadherin to inhibit neurite growth was tested in vitro. T-cadherin inhibited neurite extension from select neuron populations both as a substratum, and as a soluble recombinant protein. Anti-T-cadherin antibodies neutralized the inhibition. Spinal motor neurons were inhibited only during the stages of axon growth across the sclerotome and muscle innervation. Inhibitory responses corresponded to neuronal T-cadherin expression, suggesting a homophilic binding mechanism. These results suggest that T-cadherin is a negative guidance cue for motor axon projections.

Spectrin-actin interaction is required for neurite extension in NB 2a/dl neuroblastoma cells

Spectrin is an actin-binding membrane skeleton protein involved in the maintenance of cell shape and generation of distinct membrane protein domains. Actin binds to the N-terminal domain of beta-spectrin. To examine the function of spectrin-actin interaction in neurons, we sought to disrupt this interaction in differentiating NB 2a neuroblastoma cells by microinjecting an N-terminal domain-specific anti-beta-spectrin antibody. We found that microinjection of the affinity-purified N-terminal domain-specific anti-beta-spectrin inhibited the extension of the neurites in NB 2a/dl cells. The microinjected cells remained flat, and put out many filopodia-like processes; but these processes failed to extend when the cells were induced to differentiate in the presence of dbc AMP or in serum-free medium. The N-terminal domain-specific anti-beta-spectrin also inhibited the binding of spectrin to actin. By contrast, the microinjection of monospecific anti-alpha-spectrin(G) did not inhibit neurite extension. These results suggest that beta-spectrin-actin interaction may be required for neurite extension, which is critical for development of polarity in nerve cells.

Stable Expression and Secretion of Apolipoproteins E3 and E4 in Mouse Neuroblastoma Cells Produces Differential Effects on Neurite Outgrowth

Previously, we demonstrated in cultured dorsal root ganglion neurons that, in the presence of beta-migrating very low density lipoproteins (beta-VLDL), apolipoprotein (apo) E4, but not apoE3, suppresses neurite outgrowth. In the current studies, murine neuroblastoma cells (Neuro-2a) were stably transfected with human apoE3 or apoE4 cDNA, and the effect on neurite outgrowth was examined. The stably transfected cells secreted nanogram quantities of apoE (44-89 ng/mg of cell protein in 48 h). In the absence of lipoproteins, neurite outgrowth was similar in the apoE3- and apoE4-secreting cells. The apoE4-secreting cells, when incubated with beta-VLDL, VLDL, cerebrospinal fluid lipoproteins (d < 1.21 g/ml), or with triglyceride/phospholipid (2.7:1 (w/w)) emulsions, showed a reduction in the number of neurites/cell, a decrease in neurite branching, and an inhibition of neurite extension, whereas in the apoE3-secreting cells in the presence of a lipid source, neurite extension was increased. Uptake of beta-VLDL occurred to a similar extent in both the apoE3- and apoE4-secreting cells. With low density lipoproteins or with dimyristoylphosphatidylcholine emulsions, either alone or complexed with cholesterol, no differential effect on neurite outgrowth was observed. A slight differential effect was observed with apoE-containing high density lipoproteins. The differential effect of apoE3 and apoE4 in the presence of beta-VLDL was blocked by incubation of the cells with heparinase and chlorate, with lactoferrin, or with receptor-associated protein, all of which prevent the uptake of lipoproteins by the low density lipoprotein receptor-related protein (LRP). The data suggest that the secreted and/or cell surface-bound apoE interact with the lipoproteins and facilitate their internalization via the heparan sulfate proteoglycan-LRP pathway. The mechanism by which apoE3 and apoE4 exert differential effects on neurite outgrowth remains speculative. However, the data suggest that apoE4, which has been shown to be associated with late onset familial and sporadic Alzheimer's disease, may inhibit neuronal remodeling and contribute to the progression of the disease.

Alcohol inhibits neurite extension and increases N-Myc and c-Myc proteins

Alcohol teratogenesis may be due, in part, to inhibition of neuronal differentiation by alcohol. Because decreases in the N-myc and c-myc proteins are believed to be linked causally to neuronal differentiation, we hypothesized that alcohol would increase N-myc and c-myc proteins in undifferentiated neuronal cells and would oppose the decreases in these two proteins that normally precede differentiation. In undifferentiated LA-N-5 cultured human neuroblastoma cells, alcohol increased N-myc protein levels (178% vs. control cells) and c-myc levels (222% of control). Retinoic acid decreased N-myc and c-myc and induced neurite outgrowth (a differentiation marker). Alcohol prevented retinoic acid-elicited decreases in both myc isoforms and prevented neurite outgrowth. A significant 100% increase in c-myc and an upward trend (48%) in N-myc were observed in CA1 pyramidal neurons of the dorsal hippocampus in mouse fetuses exposed prenatally to alcohol. These data suggest that increases in N-myc and c-myc protein levels are associated with inhibition of neurite extension by alcohol.

Asymmetric retraction of growth cone filopodia following focal inactivation of calcineurin.

The neuronal growth cone is thought to be the site of decision making in nerve growth and guidance. One likely mechanism of how the growth cone translates various extracellular cues into directed motility involves rises in intracellular calcium. A variety of physiological cues, such as adhesion molecules and neurotransmitters, increases intracellular calcium, and artificial manipulations of growth cone calcium levels affect growth cone morphology and neurite outgrowth. The molecular events downstream of calcium fluxes are incompletely understood. Here we show that calcineurin, a protein phosphatase enriched in growth cones that is dependent on calcium ions and calmodulin, functions in neurite outgrowth and directed filopodial motility in cultured chick dorsal root ganglia neurons. Cyclosporin A and FK506, inhibitors of calcineurin, delayed neuritogenesis and inhibited neurite extension. Chromophore-assisted laser inactivation of calcineurin in regions of growth cones causes localized filopodial and lamellipodial retraction and influences the direction of subsequent outgrowth. We suggest that a spatial distribution of calcineurin activity within the growth cone can regulate motility and direct outgrowth.

Laminin oligopeptide derivatized agarose gels allow three‐dimensional neurite extension in vitro

The phenotypic expression of various neural cells is influenced by extracellular matrix (ECM) molecules. This study aims to develop a three-dimensional gel tailored to support neurite extension from neural cells. Laminin-derived (LN) oligopeptides CDP-GYIGSR, a 19-mer IKVAV containing sequence, GRGDSP, a cocktail of the three aforementioned LN peptides (PEPMIX), and a control peptide sequence GGGGG were covalently linked to an agarose hydrogel backbone using the bi-functional coupling agent 1'1, carbonyldiimidazole. Embryonic day 9 chick DRGs and PC12 cells were suspended in three dimensions in underivatized and derivatized agarose gels and neurite extension was analyzed. Agarose gels derivatized with CDPGYIGSR and PEPMIX enhanced neurite outgrowth from DRGs while GRGDSP and IKVAV derivatized gels inhibited neurite extension when compared to underivatized agarose gels. The IKVAV derivatized gels significantly enhanced neurite outgrowth from PC12 cells in comparison to underivatized and other LN peptide derivatized gels. Agarose hydrogels carrying covalently immobilized LN oligopeptides thus evoke specific responses from cells which contain receptors to the peptides used. Agarose hydrogels derivatized with neurite promoting peptide sequences may find applications in various models of in vivo regeneration of nervous tissue.

GAP-43 amino terminal peptides modulate growth cone morphology and neurite outgrowth

The neuronal growth-associated protein GAP-43 is expressed maximally during development and regeneration, and is enriched at the cytosolic surface of the growth cone membrane. GAP-43 can activate the GTP-binding protein G(o) which is also a major component of the growth cone membrane. These findings have led to the hypothesis that GAP-43 might modulate neurite outgrowth by altering G-protein activity. Here we define the sequence requirements for GAP-43 amino terminal peptide stimulation of G(o), and test these peptides as potential modulators of neurite outgrowth. The first 10 amino acids of GAP-43, Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln, stimulate G(o). Substitutions at particular residues reveal that cys3, cys4, arg6, and lys9 are critical, but arg7 is not. Both the GAP-43(1-10) peptide and the G-protein-activating peptide mastoparan induce growth cone collapse and inhibit neurite extension from embryonic chick dorsal root ganglion and retinal neurons. This is likely to be mediated by G-proteins: pertussis toxin blocks the inhibition, and mutant peptides that do not activate G(o) do not alter outgrowth. In contrast to the case with embryonic chick dorsal root ganglion cells, neurite outgrowth from N1E-115 neuroblastoma cells is stimulated by GAP-43(1-10). This is probably also a G-protein-mediated event because it is blocked by pertussis toxin, because the sequence requirements match those for G(o) stimulation, and because mastoparan stimulates outgrowth from these cells. The longer GAP-43(1-25) peptide does not alter neurite outgrowth unless the cells are permeabilized, suggesting an intracellular site of action. These data identify a novel set of compounds that modulate neurite outgrowth, and also support the notion that GAP-43 can alter neurite extension by modulating pertussis toxin-sensitive G-protein activity in the growth cone.

Astrocytes play a major role in the control of neuronal proliferation in vitro

The elements that control neuronal proliferation are largely unknown. Proliferating neurons in cultures of goldfish brain were studied in an attempt to identify the cell types involved. Neuronal proliferation was found to occur only when the neuronal stem cells were in direct contact with astrocytes, and never directly on the substrate. The regulation of neuronal proliferation thus appears to be mediated, at least in part, by contact with astrocytes. In addition, neurite extension was inhibited by medium conditioned by fish astrocytes. Since neurite extension and neuronal proliferation are mutually exclusive processes, inhibition of neurite extension by soluble substances derived from the astrocytes is probably one of the mechanisms controlling neuronal proliferation. The complex reciprocal relationship between neurons and astrocytes is also demonstrated by an observed inhibition of astrocytic proliferation by medium conditioned by differentiating fish neurons. This inhibition of astrocytic proliferation might be part of a mechanism through which interference with neuronal differentiation by astrocytes is avoided. The results of this study thus suggest that astrocytes, in addition to their known roles in controlling neuronal migration, neuronal differentiation and neurite elongation, may also play a role in the control of neuronal proliferation.