Establishment and neurite outgrowth properties of neonatal and adult rat olfactory bulb glial cell lines

Two glial cell types surround olfactory axons and glomeruli in the olfactory bulb (OB) and may influence synapse development and regeneration. OB astrocytes resemble type-1 astrocytes, and OB ensheathing cells resemble non-myelinating Schwann cells. We have produced clonal OB astrocyte and ensheathing cell lines from rat neonatal and adult OB cultures by SV40 large T antigen transduction. These cell lines have been characterized by morphology, growth characteristics, immunophenotype, and ability to promote neurite outgrowth in vitro. Neonatal and adult ensheathing cell lines were found to support higher neurite outgrowth than OB astrocyte lines. Neonatal OB astrocyte lines were of two types, high and low outgrowth support. The low support astrocyte lines express J1 and a chondroitin sulfate-containing proteoglycan as do astrocytes encircling the neonatal glomeruli in vivo. The adult OB astrocyte cell lines supported lower levels of outgrowth than adult ensheathing cell lines. These results are consistent with a positive role for ensheathing cells in OB synapse regeneration, in vivo. Further, based on our results, we hypothesize that ensheathing cells and high-outgrowth astrocytes facilitate axon growth in vivo, while low outgrowth astrocytes inhibit axon growth and may facilitate glomerulus formation.     

6B4 proteoglycan/phosphacan, an extracellular variant of receptor-like protein-tyrosine phosphatase zeta/RPTPbeta, binds pleiotrophin/heparin-binding growth-associated molecule (HB-GAM)

A major chondroitin sulfate proteoglycan in the brain, 6B4 proteoglycan/phosphacan, corresponds to the extracellular region of a receptor-like protein-tyrosine phosphatase, PTPzeta/RPTPbeta. Here, we purified and characterized 6B4 proteoglycan-binding proteins from rat brain. From the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) extract of brain microsomal fractions, 18-, 28-, and 40-kDa proteins were specifically isolated using 6B4 proteoglycan-Sepharose. N-terminal amino acid sequencing identified the 18-kDa protein as pleiotrophin/heparin-binding growth-associated molecule (HB-GAM). Scatchard analysis of 6B4 proteoglycan-pleiotrophin binding revealed low (Kd = 3 nM) and high (Kd = 0.25 nM) affinity binding sites. Chondroitinase ABC digestion of the proteoglycan decreased the binding affinities to a single value (Kd = 13 nM) without changing the number of binding sites. This suggested the presence of two subpopulations of the proteoglycan with different chondroitin sulfate structures. Heparin potently inhibited binding of 6B4 proteoglycan to pleiotrophin (IC50 = 3.5 ng/ml). Heparan sulfate and chondroitin sulfate C inhibited moderately (IC50 = 150 and 400 ng/ml, respectively), but, in contrast, chondroitin sulfate A and keratan sulfate were poor inhibitors (IC50 > 100 microg/ml). Immunofluorescence and immunoblotting analyses indicated that both 6B4 proteoglycan and PTPzeta are located on cortical neurons. Anti-6B4 proteoglycan antibody added to the culture medium suppressed pleiotrophin-induced neurite outgrowth of cortical neurons. These results suggested that interaction between 6B4 proteoglycan and pleiotrophin is required for the action of pleiotrophin, and chondroitin sulfate chains on 6B4 proteoglycan play regulatory roles in its binding.     

TAG-1 can mediate homophilic binding, but neurite outgrowth on TAG-1 requires an L1-like molecule and beta 1 integrins

Subsets of axons in the embryonic nervous system transiently express the glycoprotein TAG-1, a member of the subfamily of immunoglobulin (Ig)-like proteins that contain both C2 class Ig and fibronectin type III domains. TAG-1 is attached to the cell surface by a glycosylphosphatidylinositol linkage and is secreted by neurons. In vitro studies have shown that substrate-bound TAG-1 promotes neurite outgrowth. We have examined the nature of axonal receptors that mediate the neurite-outgrowth promoting properties of TAG-1. Although TAG-1 can mediate homophilic binding, neurite outgrowth on a substrate of TAG-1 does not depend on the presence of TAG-1 on the axonal surface. Instead, neurite outgrowth on TAG-1 is inhibited by polyclonal antibodies directed against L1 and, independently, by polyclonal and monoclonal antibodies against beta 1-containing integrins. These results provide evidence that TAG-1 can interact with cell surfaces in both a homophilic and heterophilic manner and suggest that neurite extension on TAG-1 requires the function of both integrins and an L1-like molecule.     

High affinity binding and overlapping localization of neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta with tenascin-R, amphoterin, and the heparin-binding growth-associated molecule

We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2-7 nM range. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by approximately 60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by approximately 75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (Kd = 0.3-8 nM), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.     

Distribution of astroglia in glomeruli of the rat main olfactory bulb: exclusion from the sensory subcompartment of neuropil

During an entire lifetime, sensory axons of regenerating olfactory receptor neurons can enter glomeruli in the olfactory bulb and establish synaptic junctions with central neurons. The role played by astrocytes in this unique permissiveness is still unclear. Glomerular astrocytes have been identified by immunocytochemistry for glial fibrillary acidic protein and S100 proteins at the light and electron microscopic levels. The latter labeling included submicroscopic lamellar and filopodial extensions of astroglial processes. Cell bodies and processes accumulate along the border between juxtaglomerular walls and glomerular neuropil. Within glomeruli, a network of astroglial processes encloses mesh-like neuropil zones devoid of astroglia. Electron microscopy confirmed the division into subcompartments of glomerular neuropil: 1) The "sensory-synaptic subcompartment" includes all sensory axon terminals and terminal dendritic branches receiving sensory input, whereas astroglia are excluded; 2) in the "central-synaptic subcompartment," astroglial processes are intermingled with other neuropil components: dendrites of relay cells and interneurons, dendrodendritic synapses, centrifugal (cholinergic and serotonergic) axons, their axodendritic synapses, and blood vessels. Unevenly distributed astroglial processes in this subcompartment are attached to vascular basal laminae, stem dendrites, and subpopulations of dendrodendritic synapses, especially those colocalized with centrifugal projections ("triadic synapses"). Astroglia-free parts of the "central" subcompartment contain segments of dendrites and subpopulations of dendrodendritic synapses. Because of the subdivision of the glomerular neuropil into portions with and without glial components, glia do not completely demarcate the border between the "sensory" and the "central" subcompartments. Interdigitation between the subcompartments varies among glomeruli and even within a single glomerulus. The mesh width of astroglial networks covaries with numerical relations between sensory and dendrodendritic synapses. This distribution pattern of astrocytes suggests that these glial cells monitor brain-derived effects on olfactory glomerular neuropil rather than olfactory input and that astroglial processes are (re-)arranged accordingly.     

Neurotrophic growth factors stimulate glycosaminoglycan synthesis in identified retinal cell populations in vitro

Glycosaminoglycans (GAG) are known to participate in central nervous system processes such as development, cell migration, and neurite outgrowth, but little is known with respect to their regulation through soluble neurotrophic factors. In the present study, we have addressed this issue using cell culture models of three distinct cell populations derived from young rat retinas, namely, purified M uller glia, pigmented epithelium, and neurons respectively. Cultures were maintained in chemically defined media in the presence or absence of either basic fibroblast or epidermal growth factor. In control glial and epithelial cultures, hyaluronic acid dominated the soluble GAG pool, with lesser contributions from dermatan sulfate, chondroitin sulfate, and heparan sulfate (in decreasing order). Retinal neuronal GAG were almost exclusively chondroitin sulfate (approximately 90%). Treatment of glial and epithelial cultures with either factor led to dose-dependent increases in especially hyaluronic acid synthesis (a maximum 6-fold increase relative to control levels), with smaller but consistent changes in chondroitin sulfate. Similar treatment of retinal neurons did not lead to any changes in GAG synthesis. These data indicate that glia and pigment epithelia are the principal sources of GAG components in retina at least in vitro, and that endogenous neurotrophic growth factors can greatly modify GAG synthesis in these two retinal cell populations. Such data suggest that a delicate balance may exist between growth factor availability and glycoconjugate metabolism in vivo, participating in normal or pathological states of the retina.     

Neurite outgrowth is enhanced by anti-idiotypic monoclonal antibodies to the ganglioside GM1

Exogenously added gangliosides enhance sprouting, neurite outgrowth, and other neuronal activities; this effect may be initiated when a ganglioside binds to a membrane protein or when a ganglioside intercalates into the plasma membrane. To test whether binding to membrane proteins is sufficient for ganglioside-mediated activity, anti-idiotypic antibodies were generated that mimic the functional binding sites of the ganglioside GM1 as described by M. J. Riggott and W. D. Matthew (1996, Glycobiology, 6, 581-589). These anti-idiotypic antibodies are proteinaceous probes that model the biochemical and biological effects of gangliosides. Those anti-idiotypic ganglioside (AIG) monoclonal antibodies (mAb's) were selected based on their ability to bind a known GM1 binding protein, the beta-subunit of cholera toxin. These studies described neuronal cell surface proteins that were identified by immunocytochemistry and Western blotting using these AIG mAb's. Here we show that AIG mAb's mimic the functional properties of GM1 in that they facilitate neurite outgrowth from central and peripheral nervous system neurons in in vitro bioassays. In addition, AIG mAb binding modulates second messenger activity, suggesting that membrane protein binding alone is sufficient to invoke intracellular activation. The similarity in the pattern of protein tyrosine phosphorylation evoked by GM1 and the anti-idiotypic ganglioside antibodies suggests that the AIG mAb's modulate neurite outgrowth in a manner similar to that of GM1. Because antibodies cannot intercalate into the plasma membrane, these results suggest that the ganglioside GM1 can mediate neuronal cellular activity by binding to cell surface proteins.     

Functional cooperation of beta1-integrins and members of the Ig superfamily in neurite outgrowth induction

Neurite outgrowth is a central aspect of the ontogenetic formation of neural networks and is regulated by distinct groups of cell surface molecules. One protein involved in neurite elongation and fasciculation is the neural Ig superfamily member F11/contactin. We have shown previously that F11 promotes neurite extension of chick tectal neurons by interaction with the tectal receptor NrCAM, a member of the L1 subgroup of the Ig superfamily. By contrast, it does not induce outgrowth of retinal neurons despite the fact that these cells also express NrCAM, suggesting that in retinal cells the F11-NrCAM interaction alone is not sufficient to induce neurite extension. In this report we present a novel image analysis procedure to quantify neurite outgrowth and use it to demonstrate that F11 enhances the fibronectin-induced outgrowth response of embryonic retinal neurons. We reveal that NrCAM is the neuronal receptor mediating the enhanced outgrowth of retinal neurons, whereas the related F11-binding molecule NgCAM is not involved. Furthermore, we provide evidence that a beta1-integrin may represent the fibronectin-dependent receptor that cooperates indirectly with the F11-NrCAM pathway. Our results support the concept of a combinatorial labeling of cells in nervous system histogenesis by different classes of cell surface proteins, in particular by integrins and molecules of the Ig superfamily.     

The core protein of the chondroitin sulfate proteoglycan phosphacan is a high-affinity ligand of fibroblast growth factor-2 and potentiates its mitogenic activity

Using a radioligand binding assay we have demonstrated that phosphacan, a chondroitin sulfate proteoglycan of nervous tissue that also represents the extracellular domain of a receptor-type protein tyrosine phosphatase, shows saturable, reversible, high-affinity binding (Kd approximately 6 nM) to fibroblast growth factor-2 (FGF-2). Binding was reduced by only approximately 35% following chondroitinase treatment of the proteoglycan, indicating that the interaction is mediated primarily through the core protein rather than the glycosaminoglycan chains. Immunocytochemical studies also showed an overlapping localization of FGF-2 and phosphacan in the developing central nervous system. At concentrations of 10 microg protein/ml, both native phosphacan and the core protein obtained by chondroitinase treatment potentiated the mitogenic effect of FGF-2 (5 ng/ml) on NIH/3T3 cells by 75-90%, which is nearly the same potentiation as that produced by heparin at an equivalent concentration. Although studies on the role of proteoglycans in mediating the binding and mitogenic effects of FGF-2 have previously focused on cell surface heparan sulfate, our results indicate that the core protein of a chondroitin sulfate proteoglycan may also regulate the access of FGF-2 to cell surface signaling receptors in nervous tissue.     

Inhibition of protein tyrosine kinases impairs axon extension in the embryonic optic tract

The role of protein tyrosine kinase (PTK) activity in the development of the retinal projection was examined in vivo by applying inhibitors of cytoplasmic PTKs, herbimycin A and lavendustin A, to intact brain preparations of Xenopus embryos. The inhibitors were present during the period when retinal ganglion cell axons first navigate through the optic tract to reach their target, the optic tectum. A majority of inhibitor-treated retinal axons stalled at the beginning of the optic tract, leading to an 80% reduction in projection length at the highest doses. All inhibitor-treated axons that did extend into the optic tract exhibited normal pathfinding behavior. Tyrosine kinase assays of inhibitor-treated brains demonstrated that at doses at which retinal axon extension was severely impaired, PTK activity, including that of src family proteins, was reduced by 50-60%. Consistent with the in vivo findings, PTK inhibitors reduced neurite outgrowth from cultured retinal neurons by 70-80%. This contrasts with the strong enhancement of outgrowth induced by the same inhibitors in cultured chick ciliary ganglion neurons and suggests that the mediation of outgrowth by PTK activity may vary in different neuronal types. Inhibitor-treated growth cones cultured on laminin were larger than normal, suggesting that tyrosine phosphorylation can modulate growth cone-substrate adhesive interactions. Our results in vivo and in vitro provide complementary evidence that retinal axon outgrowth is inhibited by pharmacological blockers of PTK activity and indicate that inhibitor-sensitive PTKs normally play a role in promoting retinal neurite extension.     

MAG and MOG enhance neurite outgrowth of embryonic mouse spinal cord neurons

Myelin-associated glycoprotein (MAG) inhibits neurite outgrowth of postnatal spinal cord neurons, but its effect on embryonic neurons is unknown. The effect on neurite outgrowth of another myelin protein, myelin-oligodendrocyte glycoprotein (MOG) is also unknown. We determined the effect of MAG and MOG on embryonic day 17 spinal cord neurons, which were cultured on MAG, MOG or control transfected CHO cells. Neurite outgrowth was examined and both total neurite length and longest neurite length were significantly enhanced by both MAG and MOG. These findings show that, in contrast to postnatal spinal cord neurons, MAG can enhance neurite outgrowth of embryonic spinal cord neurons. In addition, another myelin protein, MOG, can also modulate neurite outgrowth.     

Neural cell adhesion molecule (N-CAM) domains and intracellular signaling pathways involved in the inhibition of astrocyte proliferation

The neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferation in vitro and in vivo, and this effect is partially reversed by the glucocorticoid antagonist RU-486. The present studies have tested the hypothesis that N-CAM-mediated inhibition of astrocyte proliferation is caused by homophilic binding and involves the activation of glucocorticoid receptors. It was observed that all N-CAM Ig domains inhibited astrocyte proliferation in parallel with their ability to influence N-CAM binding. The proliferation of other N-CAM-expressing cells also was inhibited by the addition of N-CAM. In contrast, the proliferation of astrocytes from knockout mice lacking N-CAM was not inhibited by added N-CAM. These findings support the hypothesis that it is binding of soluble N-CAM to N-CAM on the astrocyte surface that leads to decreased proliferation. Signaling pathways stimulated by growth factors include activation of mitogen-activated protein (MAP) kinase. Addition of N-CAM inhibited MAP kinase activity induced by basic fibroblast growth factor in astrocytes. In accord with previous findings that RU-486 could partially prevent the proliferative effects of N-CAM, inhibition of MAP kinase activity by N-CAM was reversed by RU-486. The ability of N-CAM to inhibit astrocyte proliferation was unaffected, however, by agents that block the ability of N-CAM to promote neurite outgrowth. Together, these findings indicate that homophilic N-CAM binding leads to inhibition of astrocyte proliferation via a pathway involving the glucocorticoid receptor and that the ability of N-CAM to influence astrocyte proliferation and neurite outgrowth involves different signal pathways.     

Degradation of chondroitin sulfate proteoglycan enhances the neurite-promoting potential of spinal cord tissue

The contribution of chondroitin sulfate proteoglycan (CSPG) in the suppression of axonal growth in rat spinal cord has been examined by means of an in vitro bioassay in which regenerating neurons are grown on tissue section substrata. Dissociated embryonic chick dorsal root ganglionic neurons were grown on normal and injured adult spinal cord tissue sections treated with chondroitinases. Neuritic growth on normal spinal cord tissue was meager. However, both the percentage of neurons with neurites and the average neurite length were substantially greater on sections treated with chondroitinase ABC. Enzymes that specifically degraded dermatan sulfate or hyaluronan were ineffective. Neuritic growth was significantly greater on injured (compared to normal) spinal cord and a further dramatic increase resulted from chondroitinase ABC treatment. Neurites grew equally within white and gray matter regions after chondroitinase treatment. Observed increases in neurite outgrowth on chondroitinase-treated tissues were largely inhibited in the presence of function-blocking laminin antibodies. These findings indicate that inhibitory CSPG is widely distributed and predominant in both normal and injured spinal cord tissues. Additionally, inhibitory CSPG is implicated in negating the potential stimulatory effects of laminin that might otherwise support spinal cord regeneration.     

Ultrastructural localization of P2X3 receptors in rat sensory neurons

We used isolated IgG antibodies selective for P2X3 receptors to study the ultrastructural distribution of these receptors in rat sensory neurons. In trigeminal ganglia, P2X3 receptor immunoreactivity occurred in small and large nerve cell bodies and their processes. Endoplasmic reticulum and Golgi apparatus were heavily stained; cytoplasmic matrix was faintly to moderately stained. In synaptic glomeruli in lamina II of cervical dorsal horn, P2X3 receptor-immunoreactive core terminals were postsynaptic to unlabelled vesicle-containing dendrites and axons. In the nucleus of the solitary tract, receptor-positive boutons synapsed on dendrites and cell bodies and had complex synaptic relationships with other axon terminals and vesiculated dendrites. These observations identify sites from which ATP could be released to influence sensory signalling within the central nervous system.     

Basic fibroblast growth factor enhances the growth of postnatal neostriatal GABAergic neurons in vitro

Basic fibroblast growth factor (bFGF) significantly enhances the short-term survival of embryonic striatal neurons in vitro but has little effect on the outgrowth of striatal cells compared to neurons from other brain regions. Studies in our laboratory have shown that bFGF protects postnatal striatal cells in vitro from NMDA receptor-induced neurotoxicity. We therefore examined the effects of bFGF on the outgrowth of GABA-containing cells taken from the postnatal (Day 1) caudate-putamen and cultured for up to 3 weeks. In control cultures GABAergic neurons formed three populations based on somatic size and developed the cytoarchitectural features characteristic of dendrites, spines, and axons. In the presence of bFGF (6 pM continuously from the day of plating), small- and medium-sized GABAergic neurons showed significant increases compared to untreated controls in axon-like growth (axon length) at 6 days in culture and in both axon- and dendrite-like neurite growth (axon length and branch order, number of primary dendrites, dendrite length, and dendritic branch order) at 13 and 17 days in culture. Large GABAergic neurons were unaffected by treatment with bFGF. Striatal GABAergic neurons exposed to nerve growth factor (10 ng/ml) were not different from untreated controls. Neuron survival was also unaffected by bFGF treatment at all days in culture examined. Other observations suggested that the neurotrophic effects of bFGF were mediated by a direct action of the growth factor on striatal neurons and not glial cells. First, glial cells (identified by the immunohistochemical localization of glial fibrillary acidic protein) were unaffected by bFGF treatment at the low concentration (6 pM) used to enhance neurite growth, but did significantly proliferate at higher concentrations of bFGF (6 nM). Second, immunoreactive bFGF receptor protein was localized predominantly to the somata and processes of striatal neurons and not to glial cells in the cultures. Finally, when neurons from control cultures were briefly exposed (1 to 4 h) to bFGF at concentrations which were neurotrophic, a marked elevation in the immediate early gene protein c-fos was observed by immunohistochemistry in the nuclei of neurons, including GABAergic cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

A functional interaction between the neuronal adhesion molecules TAG-1 and F3 modulates neurite outgrowth and fasciculation of cerebellar granule cells

F3 and TAG-1 are two closely related adhesion glycoproteins of the Ig superfamily that are both expressed by the axons of cerebellar granule cells. In an in vitro system in which cerebellar granule cells were cultured on monolayers of transfected Chinese hamster ovary (CHO) cells, we show that F3 and TAG-1 interact functionally. F3 transfectants have been shown to inhibit outgrowth and induce fasciculation of granule cell neurites. By contrast TAG-1 transfectants have no effect on these events. However, when TAG-1 is coexpressed with F3, the inhibitory effect of F3 is blocked. Two possible mechanisms may account for this functional interaction: (1) either TAG-1 and F3 compete for the same neuronal receptor, and in favor of this we observed that binding sites for microspheres conjugated with F3 and TAG-1 are colocalized on the granule cell growth cones, (2) or alternatively, F3 and TAG-1 associate in a multimolecular complex after their binding to independent receptors. Extensive co-clustering of F3 with TAG-1 can in fact be achieved by anti-TAG-1 antibody-mediated cross-linking in double-transfected CHO cells. Moreover, F3 coimmunoprecipitates with TAG-1 in Triton X-100-insoluble microdomains purified from newborn brain. These data strongly suggest that F3 and TAG-1 may associate under physiological conditions to modulate neurite outgrowth and fasciculation of the cerebellar granule cells.     

Rac1-dependent actin filament organization in growth cones is necessary for beta1-integrin-mediated advance but not for growth on poly-D-lysine

The activity of filopodia and lamellipodia determines the advance, motility, adhesion, and sensory capacity of neuronal growth cones. The shape and dynamics of these highly motile structures originate from the continuous reorganization of the actin cytoskeleton in response to extracellular signals. The small GTPases, Rac1, Rho, and CDC42, regulate the organization of actin filament structures in nonneuronal cells; yet, their role in growth cone motility and neurite outgrowth is poorly understood. We investigated in vitro the function of Rac1 in neurite outgrowth and differentiation by introducing purified recombinant mutants of Rac1 into primary chick embryo motor neurons via trituration. Endogenous Rac1 was expressed in growth cone bodies as well as in the tips and shafts of filopodia, where it often colocalized with actin filament structures. The introduction of constitutively active Rac1 resulted in an increase in rhodamine-phalloidin staining, presumably from an accumulation of actin filaments in growth cones, while dominant negative Rac1 caused a decrease in rhodamine-phalloidin staining. Nevertheless, both Rac1 mutants retarded growth cone advance, and hence attenuated neurite outgrowth and inhibited differentiation of neurites into axons and dendrites on laminin and fibronectin. In contrast, on poly-D-lysine, neither Rac1 mutant affected growth cone advance, neurite outgrowth, or neurite differentiation despite inducing similar changes in the amount of rhodamine-phalloidin staining in growth cones. Our data demonstrate that Rac1 regulates actin filament organization in neuronal growth cones and is pivotal for beta1 integrin-mediated growth cone advance, but not for growth on poly-D-lysine.     

Plastic changes in Ara C-treated and "transplanted" coeruleocerebellar cultures

Locus coeruleus axons project to cerebellar cortex in coeruleocerebellar cultures, where they make functional contacts, and also appear as fine fibers in the outgrowth zones. The predominant catecholamine of locus coeruleus neurons in culture is dopamine. When coeruleocerebellar cultures are exposed to cytosine arabinoside to destroy cerebellar granule cells and functionally compromise glia, there is a resultant increase of Purkinje cell survival and a sprouting of Purkinje cell recurrent axon collaterals, plus an increase of catecholaminergic axons accompanied by a doubling of tissue dopamine content. If such reorganized cultures are transplanted with granule cells and glia, a second round of plastic changes ensues in which the Purkinje cell population and the recurrent axon collaterals are reduced to control levels, but catecholaminergic axons and dopamine content remain increased. The maintenance of catecholaminergic axons does not appear to depend on the persistence of target neurons.     

Syntaxin and 25-kDa synaptosomal-associated protein: differential effects of botulinum neurotoxins C1 and A on neuronal survival

The Clostridium botulinum neurotoxins (BoNTs) A and C1 cleave specific proteins required for neuroexocytosis. We demonstrated that, in intact neurons, BoNT A cleaves 25-kDa synaptosomal-associated protein (SNAP-25), and BoNT C1 cleaves both syntaxin and SNAP-25 (Williamson et al.: Mol Biol Cell 6:61a, 1995; J Biol Chem 271:7694-7699, 1996). Here, we compare the actions of BoNT A and BoNT C1 on mature and developing mouse spinal cord neurons in cell culture and demonstrate that BoNT C1 is severely neurotoxic. In mature cultures, synaptic terminals become enlarged shortly after BoNT C1 exposure, and, subsequently, axons, dendrites, and cell bodies degenerate. Electron microscopy confirms that early degenerative changes occur in synaptic terminals when the somatic cytoplasm appears normal. In newly plated cultures, few neurons survive exposure to BoNT C1. Whereas both BoNT A and BoNT C1 cleave SNAP-25, BoNT A has no adverse effect on neurite outgrowth, synaptogenesis, or neuron survival. This cytotoxicity is unique to BoNT C1, is specific to neurons, and is initiated at the synaptic terminal, suggesting either a novel role for syntaxin or additional actions of BoNT C1. The neurodegeneration induced by BoNT C1 may be significant in terms of its efficacy for the clinical treatment of dystonia and spasticity.