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PubMed2006

CyclinB2 and BIRC5 genes as surrogate biomarkers for neurite outgrowth in SH-SY5Y subclonal cells.

Neurite outgrowth plays a key role in neuronal development and regeneration, and is the hallmark assay for the effects of neurotrophic factors such as nerve growth factor (NGF). However, measuring neurite outgrowth is a slow and resource-intensive process. We therefore wanted to identify surrogate biomarkers for neurite outgrowth activity by gene expression analysis in SH-O10 cells, a subclone of the human SH-SY5Y neuroblastoma cell line but with much higher NGF-induced neurite outgrowth activity. Microarray analysis identified seven genes where mRNA levels were changed. NGF-induced decreases in levels of two genes, CyclinB2 and BIRC5, were confirmed by quantitative real-time RT-PCR. Levels of NGF-induced decreases in CyclinB2 and BIRC5 mRNA in several SH-SY5Y subclones with different neurite outgrowth responses correlated with their neurite outgrowth activities. Decreases in CyclinB2 and BIRC5 mRNA induced by FK506 or retinoic acid, both of which exert potentiation of NGF-induced neurite outgrowth effects but with different mechanisms, also correlated with their neurite outgrowth activities. In conclusion, decreasing levels of CyclinB2 and BIRC5 mRNA strongly correlate with neurite outgrowth activities in terms of NGF-related effect in SH-SY5Y subclonal cells, and have potential to become quantitative surrogate biomarkers for measuring NGF-related neurite outgrowth.

Antineoplastic Agents↗
PubMed1998

Activation of mitogen-activated protein kinases is not required for the extension of neurites from PC12D cells triggered by nerve growth factor.

Numerous studies with PC12 cells have suggested that the mitogen-activated protein (MAP) kinase pathway might play a major role in the neuronal differentiation that is induced by nerve growth factor (NGF). Cells of the PC12D subline extend neurites within several hours in response to NGF in the presence of inhibitors of the synthesis of RNA and protein. We examined the effects of a specific inhibitor 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059) of the MAP kinase kinase (MEK)/MAP kinase pathway on the NGF-induced outgrowth of neurites in PC12D cells. The increase in MAP kinase activity in response to NGF was reduced by 80% upon treatment of PC12D cells with 50 microM PD98059, whereas the NGF-dependent formation of ruffles and the subsequent outgrowth of neurites were not blocked by PD98059 at this concentration. The outgrowth of neurites from conventional PC12 cells by NGF was suppressed by the addition of 50 microM PD98059 as reported by Pang et al. [L. Pang, T. Sawada, J. Stuart,S.J. Decker, A.R. Saltiel, Inhibition of MAP kinase kinase blocks the differentiation of PC12 cells induced by nerve growth factor, J. Biol. Chem. 270 (1995) 13585-13588]. In contrast, the rapid regeneration of neurites from PC12 cells primed with NGF, was not altered in the presence of the same dose of the inhibitor of MEK. It appeared, therefore, that the activation of the MAP kinase pathway was not necessarily required for the NGF-dependent extension of neurites. When PC12D cells were transfected with the dominant inhibitory Ha-ras Asn-17 gene, the induction of the mutant Ras protein led the suppression of the rapid outgrowth of neurites in response to NGF but not to dibutyryl cyclic AMP (dbcAMP). The result implies a direct involvement of Ras protein in the NGF-induced signal transduction that lead to the formation of neurites in PC12D cells. We can conclude that the activation of MAP kinase and selective gene expression are required for the differentiation of conventional PC12 cells to sympathetic neuron-like cells and that activation of Ras protein and, subsequently, of a MAP kinase-independent pathway might be involved in the extension of neurites from PC12D cells or in the regeneration of neurites from primed PC12 cells in response to NGF.

Animals↗
PubMed1999

A rapid method for semi-quantitative analysis of neurite outgrowth from chick DRG explants using image analysis.

Neurite outgrowth from dorsal root ganglion (DRG) explants is a method of evaluating neurotrophic activity of growth factors and neurotrophin mimetics. The drawbacks to this approach are the difficulties in quantifying the response. Neurite counts are time consuming and labour intensive, and the accuracy is often questionable due to branching and fasciculation of the neurites. We report here a method of semi-quantitative analysis of neurite outgrowth from chick DRG explants, using image analysis to quantify the area occupied by neurites emanating from the ganglion. This method is rapid, takes into account both the length and number of neurites, and is unaffected by neurite fasciculation or branching. Primary explants of chick DRGs were treated with the neurotrophins nerve growth factor (NGF) or neurotrophin-3 (NT-3) and with the compound K252a. K252b was tested for potentiation of the response to NT-3. The results show a dose dependent outgrowth of neurites from explants treated with NGF, NT-3 and K252a, and potentiation of the NT-3 response by K252b. These responses were quantified by neurite area quantification using image analysis. We conclude that neurite area measurement using image analysis provides a robust means of evaluating neurotrophic activity of growth factors and neurotrophin mimetics in vitro.

Animals↗
PubMed2003

Concentration-dependent actions of glial chondroitin sulfate on the neuritic growth of midbrain neurons.

Astrocytes located in two distinct regions of midbrain differ in their neuritic growth support abilities. Midbrain neurons cultured onto astrocyte monolayers from the lateral (L) region develop long and branched neurites while neurons cultured onto astrocyte monolayers from the medial (M) region develop short or no neurites. The extracellular matrix of these astrocytes has an important role in promoting or inhibiting the growth of these neurons. Differences on the compartmental distribution, as well as on the concentration of GAGs of L and M astrocytes, may be related to their differential capacity of supporting neuritic growth. Indeed, enzymatic digestion of heparan sulfate (HS) and chondroitin sulfate (CS) chains also pointed to an important function for GAGs on axon navigation. In order to better characterize the role of CS on the growth of midbrain neurites, we treated L and M astrocyte monolayers with 1 mM of beta-D-xyloside. Under these conditions, astrocytes oversynthesized and secreted CS protein-free chains to the culture medium. M astrocytes had a significant reduction in their neuritic growth-inhibiting ability after xyloside treatment, suggesting a promoting role for soluble CS in neuritic growth. Chondroitin 4-sulfate (CS-4) added in different concentrations to M astrocyte cultures turned this glia into a permissive substrate, acting in a linear way as far as the largest neurite was concerned. However, a U-shaped dose-effect curve on neurite growth resulted from the similar treatment of L astrocytes. These results suggest that glial CS-4 could be involved in the neurite growth modulating properties of midbrain neurons in a complex concentration-dependent way.

Animals↗
PubMed2002

L- and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) inhibit neurite outgrowth from SH-SY5Y cells.

Gangliosides and extracellular matrix molecules influence neurite outgrowth, but the combinatorial effects of these endogenous agents on outgrowth are unclear. Exogenous gangliosides inhibit neurite outgrowth from SH-SY5Y cells stimulated with platelet-derived growth factor-BB, and different isoforms of the ceramide analog threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) stimulate (L-PDMP) or inhibit (D-PDMP) glycosphingolipid biosynthesis. In this study, we determined whether altering the endogenous ganglioside levels with PDMP in SH-SY5Y cells regulates neurite outgrowth on the outgrowth-supporting extracellular matrix molecule, laminin. In cells stimulated with 20 ng/ml platelet-derived growth factor-BB to promote outgrowth, we used image analysis to evaluate neurite outgrowth from SH-SY5Y cells grown on endogenous matrix or laminin and exposed to L- or D-PDMP. Both L- and D-PDMP decreased neurite initiation (the number of neurites/cell, the percent of neurite-bearing cells), elongation (the length of the longest neurite/cell, the total neurite length/cell), and branching (the number of branch points/neurite) from SH-SY5Y cells on endogenous matrix or laminin in a dose-dependent manner in serum-free or serum-containing medium. The inhibitory effects of each PDMP isoform were reversible. Inhibition of neurite outgrowth by L-PDMP could be mimicked by addition of exogenous gangliosides or C2-ceramide. Our analyses of neurite outgrowth in SH-SY5Y cells, a model of developing or regenerating noradrenergic neurons, demonstrate that increasing or decreasing endogenous ganglioside levels decreases neurite outgrowth. These results may indicate that SH-SY5Y cells undergo tight regulation by gangliosides, possibly through modulation of growth/trophic factor- and/or extracellular matrix-activated signaling cascades.

Gangliosides↗
PubMed1998

Brain-derived neurotrophic factor stimulates neurite outgrowth in a calretinin-enriched neuronal culture system.

A calretinin enriched cell culture system which comprised approximately 40% of the total neuronal population of the E14 rat embryo was established from the region of the thalamic eminence (TE), and the effects of several neurotrophins on the neurite growth of calretinin-immunoreactive (CR-IR) neurons was investigated. A 4-day treatment of BDNF significantly increased the ratio of CR-IR to microtubule-associated protein 2-immunoreactive neurons at concentrations between 50 and 250 ng/ml. IGF-I at 100 ng/ml and TGF-alpha at 250 ng/ml also increased this ratio. None of the neurotrophins examined increased the number of primary neurites. BDNF did, however, increase the number of secondary neurites. BDNF-treated primary and secondary neurites were also significantly longer than neurites from neurons in control cultures. IGF-I elicited an increase in primary neurite length, but did not affect either number or length of secondary neurites. TGF-alpha had no effect on either number or length of the primary and secondary neurites. These results indicate that the maturation and development of CR-IR neurites is specifically affected by BDNF. It is suggested that BDNF increases the CR concentration above the threshold of detection by immunohistochemistry in cells and stimulates the sprouting of secondary CR-IR neurites.

Animals↗
PubMed1995

Enhancement of neurite outgrowth following calpain inhibition is mediated by protein kinase C.

We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell-permeant cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal ("C1") and calpeptin or by the phospholipid-mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule-associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2-O-tetradecanoylphorbol 13-acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down-regulation following TPA treatment. Cell-free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium-requiring calpain to cleave the SH-SY-5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity.

Blood Physiological Phenomena↗
PubMed2005

Identification of conserved amino acids N-terminal of the PKC epsilon C1b domain crucial for protein kinase C epsilon-mediated induction of neurite outgrowth.

We have shown previously that protein kinase C (PKC) epsilon can induce neurite outgrowth independently of its catalytic activity via a region encompassing its C1 domains. In this study we aimed at identifying specific amino acids in this region crucial for induction of neurite outgrowth. Deletion studies demonstrated that only 4 amino acids N-terminal and 20 residues C-terminal of the C1 domains are necessary for neurite induction. The corresponding regions from all other novel isoforms but not from PKCalpha were also neuritogenic. Further mutation studies indicated that amino acids immediately N-terminal of the C1a domain are important for plasma membrane localization and thereby for neurite induction. Addition of phorbol ester made this construct neurite-inducing. However, mutation of amino acids flanking the C1b domain reduced the neurite-inducing capacity even in the presence of phorbol esters. Sequence alignment highlighted an 8-amino acid-long sequence N-terminal of the C1b domain that is conserved in all novel PKC isoforms. Specifically, we found that mutations of either Phe-237, Val-239, or Met-241 in PKCepsilon completely abolished the neurite-inducing capacity of PKCepsilon C1 domains. Phorbol ester treatment could not restore neurite induction but led to a plasma membrane translocation. Furthermore, if 12 amino acids were included N-terminal of the C1b domain, the C1a domain was dispensable for neurite induction. In conclusion, we have identified a highly conserved sequence N-terminal of the C1b domain that is crucial for neurite induction by PKCepsilon, indicating that this motif may be critical for some morphological effects of PKC.

Amino Acid Sequence↗
PubMed1995

Exocytosis and selective neurite calcium responses in rat cerebellar granule cells during field stimulation.

The free calcium concentration, [Ca2+]c, in fura-2-loaded rat cerebellar granule cells was investigated by digital imaging during trains of uniform field stimuli in order to compare the ability of calcium channels in somata and neurites to respond to brief, physiologically relevant depolarizations. Very few somata responded to 20 Hz trains of 1 ms pulses, while virtually all neurites showed an extensive increase which was rapidly reversed when stimulation was terminated. In contrast, both somata and neurites responded when cells were depolarized with 50 mM KCI. The field stimuli evoked a tetrodotoxin-sensitive increase in Na+ concentration in both somata and neurites. When 4-aminopyridine, which inhibits delayed K+ currents in these cells, was present during the field stimulus both somata and neurites increased their [Ca2+]c, suggesting that prolongation of the duration of depolarization is required for somatic Ca2+ channel activation. The neurite response did not depend on the orientation of the neurite relative to the applied field. The neurite response was insensitive to nifedipine (1 microM) and omega-agatoxin-IVA (30 nM) but was uniformly inhibited by omega-conotoxin-GVIA (30% inhibition at 1 microM) and omega-conotoxin-MVIIC (44% inhibition at 5 microM). The two inhibitors were not additive. The neurite [Ca2+]c response was insensitive to the combination of ionotropic glutamate receptor antagonists. Field stimulation caused the exocytosis of the fluorescent probe FM1-43 previously loaded during KCI depolarization, suggesting that presynaptic Ca2+ channels contribute to the field-evoked neurite response.

Animals↗
PubMed2000

Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells.

The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.

Animals↗
PubMed1991

Assembly of actin-containing cortex occurs at distal regions of growing neurites in PC12 cells.

Although actin filaments are known to be localized in the cortex of axons and in the growth cones of nerve cells, it is unclear how actin-containing structures are assembled during nerve growth. We have studied the formation of actin structures in growing neurites by microinjecting fluorescent phalloidin or actin into PC12 neuron-like cells to label endogenous actin filaments. Upon stimulation of neurite growth in cells microinjected with fluorescent phalloidin, little or no fluorescence was detected in nascent growth cones and adjacent neurites despite the presence of actin filaments in these regions, suggesting that actin filaments were primarily formed by de novo assembly rather than the transport and reorganization of pre-existing, phalloidin-labeled actin filaments. Time-lapse observations of the distribution of phalloidin-labeled actin filaments during neurite elongation confirmed that fluorescence associated with pre-existing neurite cortex spread out more slowly than the elongation of neurites. Furthermore, when a dark spot was photobleached with a laser microbeam along neurites of cells microinjected with either fluorescent phalloidin or actin, the spot showed no appreciable translocation during active neurite elongation. Taken together, these results suggest that de novo assembly of actin filaments plays a crucial role in the formation of growth cones and adjacent cortex in the distal region of neurites, but does not appear to require the anterograde or retrograde transport of cortical filaments, or the passive stretching of the proximal segment of the neurite cortex.

Actins↗
PubMed1993

Dual function of tenascin: simultaneous promotion of neurite growth and inhibition of glial migration.

The extracellular matrix molecule tenascin is expressed within the developing peripheral nervous system, first by migrating neural crest cells and later by satellite (Schwann precursor) cells at the growing tips of peripheral nerves. Here we found that the neurite promoting activity of tenascin for sensory neurons is developmentally regulated: very young sensory ganglia of stage 23 (4 days old) embryos grew neurites on tenascin as fast as on laminin and fibronectin. The growth response of older (day 7 and 9) ganglia on laminin and fibronectin was similar to that of 4-day-old ganglia, while on tenascin neurite growth occurred only after a lag phase and at a slower rate. Neurite growth on tenascin was inhibited by antibodies to beta 1 integrin and by heparin. While tenascin promotes neurite outgrowth of peripheral neurons, we found that it does not allow satellite cell migration when it is present on the substratum, and it inhibits migration of satellite cells on fibronectin when added in soluble form. In contrast, soluble tenascin did not significantly alter the rate of neurite growth on tenascin, fibronectin or laminin substrata, although neurites were straighter and less attached. When isolated satellite cells were added to neurites grown on tenascin, they preferentially adhered to and elongated along neurite surfaces. Using patterned substrata of tenascin versus fibronectin or laminin confirmed that tenascin borders allow neurites to pass but act as barriers to migrating satellite cells. We postulate that tenascin or related molecules with dual functions in cell adhesion are important for peripheral nerve morphogenesis. Tenascin allows axonal growth, but may restrict random satellite cell migration into the fibronectin-rich mesenchyme, thereby inducing the compaction of nerve fascicles.

Animals↗
PubMed1995

Overexpressing cell surface beta 1.4-galactosyltransferase in PC12 cells increases neurite outgrowth on laminin.

Neurite outgrowth on cellular and extracellular matrices is mediated by a variety of cell surface receptors. Some of these receptors recognize peptide determinants, whereas others bind oligosaccharide ligands. Previous studies have suggested that cell surface beta 1.4-galactosyltransferase functions as one of these receptors during neurite outgrowth on basal lamina by binding to N-linked oligosaccharides in the E8 domain of laminin. However, these previous investigations have been limited to the use of galactosyltransferase inhibitory reagents to block neurite formation. Therefore, in this study, we investigated whether the level of surface galactosyltransferase directly affects the efficiency of neurite outgrowth, or rather, is incidental to neurite formation. Northern blot analysis and cell surface galactosyltransferase assays were used to select two stable PC12 transfectants that overexpress surface galactosyltransferase by approximately four-fold. Radiolabeled antibody binding to intact cells and indirect immunofluorescence confirmed the higher expression of surface galactosyltransferase on transfected cells, compared to controls. Both galactosyltransferase transfected cell lines exhibited markedly enhanced neurite initiation, neurite formation, and rates of neurite elongation by two- to three-fold. These studies demonstrate that the expression of laminin receptors can be rate-limiting during neurite outgrowth, and that the level of surface galactosyltransferase can modulate the frequency and rate of neurite formation from PC12 cells on laminin.

Animals↗
PubMed1998

Soluble factors from rat basophilic leukemia (RBL-2H3) cells stimulated cooperatively the neurite outgrowth of PC12 cells.

Culture media from rat basophilic leukemia cells (RBL-2H3) induced the neurite outgrowth of rat pheochromocytoma PC12 cells, a model system for neuronal differentiation. The extension of the neurite outgrowth was dependent on the culture time of RBL-2H3 cells in the DMEM medium. The DMEM medium conditioned by RBL-2H3 cells for 48 h induced neurite outgrowth of PC12 cells significantly. The neurite extension was much higher than that by medium containing 1 ng/ml nerve growth factor (NGF) but was rather lower than that by medium containing 10 or 50 ng/ml NGF. The neurite extension by 50 ng/ml NGF was completely suppressed by excess anti-NGF antibody (1-1.5 microg/ml), while the extension by culture medium conditioned by RBL-2H3 cells for 48 h was not completely suppressed in the presence of the same amount of anti-NGF antibody. The neurite extension by the culture medium of RBL-2H3 cells was also suppressed by anti-interleukin (IL)-6 antibody (1 microg/ml), although IL-6 itself (20 units) could scarcely induce the neurite outgrowth of PC12 cells. This suggests that IL-6 in the culture medium of RBL-2H3 cells could be effective in inducing the neurite extension in cooperation with NGF. In the presence of an excess of both anti-NGF and anti-IL-6 antibodies, the culture medium of RBL-2H3 cells induced the neurite extension of PC12 cells. This suggests that the action of the various factors from RBL-2H3 cells may be synergistic as far as the neurite outgrowth of PC12 cells is concerned.

Animals↗
PubMed1995

Maturation-dependent upregulation of growth-promoting molecules in developing cortical plate controls thalamic and cortical neurite growth.

We have tested the hypothesis that maturation-dependent changes in the cortical plate affect the spatiotemporal growth patterns of developing thalamocortical and corticocortical axonal projections. Given a choice between alternating lanes of embryonic (E18-19) and neonatal (P0-1) rat cortical plate membranes, embryonic (E18-19) thalamic and cortical neurites prefer to extend on neonatal membranes. Thalamic and cortical explants do extend neurites on uniform carpets of E19 cortical plate membranes, but the outgrowth is consistently greater on uniform carpets of P1 cortical plate membranes. These experiments demonstrate a maturation-dependent enhancement in the ability of cortical plate to support neurite growth from thalamic and cortical explants. In contrast, retinal and cerebellar neurites, which do not grow into cortex in vivo, generally grew poorly on these membranes, suggesting a degree of specificity to the neurite growth response. Immunohistochemical analysis of developing cortex suggests that several extracellular matrix (ECM) and cell adhesion molecules are upregulated in cortical plate. However, immunocharacterization of membrane carpets for these same ECM and cell adhesion molecules suggests that the growth preferences of thalamic and cortical neurites in vitro are predominantly influenced by membrane-anchored, rather than ECM, molecules. Western analysis of E19 and P1 cortical plate membranes supports this conclusion, and indicates that the membrane-anchored cell adhesion molecules L1 and N-CAM are more abundant in the P1 cortical plate membrane preparation. Experiments in which cortical plate membranes were treated to remove molecules sensitive to phosphatidylinositol (PI)-specific phospholipase C demonstrate that neurite growth promoters present in E19 cortical plate membranes are predominantly PI linked, whereas those present in P1 membranes are predominantly non-PI linked. These findings indicate that the neurite growth preferences are mediated, at least in part, by an upregulation of neurite growth-promoting molecules in developing cortical plate that are not PI linked. Taken together, these findings suggest that a maturation-dependent upregulation of neurite growth-promoting molecules on cortical plate cells controls the invasion of the cortical plate by thalamocortical and corticocortical axons.

Aging↗
PubMed1995

Cholinergic regulation of neurite outgrowth from isolated chick sympathetic neurons in culture.

Neurotransmitters have been reported to regulate neurite outgrowth in several vertebrate and nonvertebrate species. In this study, cultures of isolated embryonic day 12 (E12) chick sympathetic neurons were grown in the presence of cholinergic receptor agonists or antagonists. Both ACh and the nonhydrolyzable cholinergic agonist carbamylcholine (CCh) inhibited neurite outgrowth. ACh (0.1-1.0 mM) decreased the percentage of neurons bearing neurites, but had no significant effect on cell survival. The effect of ACh was increased in the presence of the cholinesterase inhibitors BW284C51 (1 microM), Tacrine (20 microM), and edrophonium (200 microM). Neurite outgrowth was strongly inhibited by the muscarinic receptor agonist oxotremorine (5-100 microM) and weakly inhibited by nicotine (50 nM to 10 microM). The inhibitory effect of CCh was decreased by the muscarinic receptor antagonist atropine (10 microM), demonstrating that the effect of CCh on neurite outgrowth was mediated, at least in part, through a muscarinic receptor. The possibility that AChE can influence neurite outgrowth directly, through a noncatalytic mechanism, was also examined. When dissociated chick brain or sympathetic neurons were grown on plates precoated with purified AChE, neurite outgrowth was strongly stimulated. However, the neurite outgrowth-promoting effect of AChE was strictly dependent upon the presence of substratum-bound heparan sulfate proteoglycans (HSPG). Pretreatment of AChE with diisopropylfluorophosphate to inhibit the esterase activity did not abolish this effect, suggesting that the neurite outgrowth-promoting effect of AChE was associated with a noncatalytic mechanism, a view supported by the observation that soluble AChE had no effect on neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1994

Astroglial differentiation is required for support of neurite outgrowth.

Models of astrocyte differentiation stress a lineage program that involves a progressive loss of astroglial support of neuronal differentiation. These models predict that astroglial promotion of neurite extension declines with the "age" of the astrocyte. An alternative view is that astroglial support of neurite growth is regulated by epigenetic factors that induce the cells either to differentiate and support neuronal functions or to undergo cell proliferation and fail to support neurons. To compare the contribution of astroglial cell "age" to astroglial support of neurite extension, mouse cerebellar astroglia were maintained in vitro for 3-90 d, and assayed for their ability to support neurite formation. When cultured in isolation, astroglial support of neurite extension declined with time in vitro, as assayed by quantifying outgrowth from explants of pontine nuclei, falling from a robust level just after the astroglia were harvested to negligible levels 21-90 d later. Since previous studies have shown that neurons can change the state of astroglial cells (Hatten, 1985), we tested the neurite promoting activity of astroglia that were cultured for 21-90 d in vitro and subsequently induced to differentiate by the addition of neurons. When granule neurons were added to aged astroglia and pontine explants plated 2 d later, neurite growth from the explants was exuberant, regardless of the time astroglia spent in vitro prior to the addition of neurons. The state of astroglia that were growth promoting or growth inhibiting was examined by bromodeoxyuridine staining and with antisera to glial filament protein. Aged astroglia cultured alone and thus inhibitory to axon growth, proliferated at high rates and had polygonal shapes. In contrast, aged astroglia to which neurons had been added, proliferated at low rates and developed process-bearing stellate shapes. To test further whether proliferation levels related to the growth-supporting properties of astroglia, astroglia were plated alone in medium without serum, or with the addition of transforming growth factor-beta 1, each treatment known to arrest proliferation. In both cases, promotion of neurite growth was restored in aged astroglia, but the morphology of astroglia did not correlate with the ability to support neurite growth. Finally, the growth-inhibiting properties of aged astroglia do not appear to be mediated by diffusible factors, and require close apposition with living astroglial cells. We conclude that astroglial support of neurite extension depends on the state of differentiation of astroglial cells, and that these properties can be modified by coculture with neurons or conditions that arrest of astroglial proliferation, irrespective of astroglial "age".

Animals↗
PubMed1994

Neuritic deposition of agrin on culture substrate: implications for nerve-muscle synaptogenesis.

Recent experiments have indicated that neural agrin is deposited at newly forming nerve-muscle synapses and has a primary synaptogenic role there. As a step toward assessing how the spatial arrangement of new synaptic sites is regulated, we compared the pattern of agrin deposition by Xenopus neurites on culture substrate and on muscle cells. The neurons were grown on a substrate that bound their externalized agrin so tightly that it remained bound even when the neurites retracted spontaneously or were eliminated experimentally. By contrast, the neural cell adhesion molecule, NCAM, was not left behind on the substrate when the neurites were eliminated. Agrin, visualized by immunofluorescent staining, was deposited on the culture substrate in a continuous fashion along virtually the entire neuritic arbor of many spinal cord (SC) neurites. The pattern of agrin deposition by the same neurites changed from continuous to discontinuous when the neurites contacted muscle cells, and it became continuous again when the neurites returned to the culture substrate. The sites of agrin deposition on muscle cells were also sites of accumulation of ACh receptors (AChRs). Dorsal root ganglion (DRG) neurons and some SC neurons did not deposit agrin along their neuritic outgrowth, either on the culture substrate or on the muscle cells, and did not induce AChR accumulation at sites of contact with muscle cells. Besides adding to the evidence in support of agrin's synaptogenic role, the findings indicate that muscle cells significantly influence how neural agrin and synaptic sites become distributed along paths of neurite-muscle contact.

Agrin↗