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J L Bixby

Publications and source records attributed to J L Bixby.

At least 37 records · Page 2Linked to original sources
PubMed1996

Intracellular mechanisms of axon growth induction by CAMs and integrins: some unresolved issues.

Integrins and cell adhesion molecules (CAMs) are important neuronal receptors mediating substrate-induced axon growth. Signaling of axon growth through these receptors involves both regulation of tyrosine phosphorylation and transient increases in intracellular Ca2+. Many of the details concerning these signal transduction events and mechanisms through which they regulate effectors of axon growth are poorly understood. This review discusses some of the gaps in our current knowledge, with suggestions on approaches to closing these gaps. Emphasis is on the role of tyrosine phosphatases in the regulation of axon growth, the origin and nature of Ca2+ signals produced by stimulation of CAMs and integrins, and possible links of these two pathways to cytoskeletal rearrangements and directed addition of plasma membrane.

Animals↗
PubMed1995

Collagen synthesis inhibition reduces clustering of heparan sulfate proteoglycan and acetylcholine receptors but not agrin or p65, at neuromuscular contacts in vitro.

We have studied presynaptic and postsynaptic differentiation at neuromuscular junctions in vitro by examining the localization of synapse-specific proteins. In nerve-muscle co-cultures, the synaptic vesicle protein synaptotagmin (p65) accumulated in the nerve terminal overlying myotubes in association with postsynaptic clusters of acetylcholine receptors (AChRs), heparan sulfate proteoglycan (HSPG), laminin, and agrin. Inhibition of collagen synthesis with cis-hydroxyproline decreased the nerve-induced clustering of AChRs in muscle cells as well as that caused by exogenous agrin in muscle-only cultures. Moreover, accumulation of HSPG at contacts was also inhibited in cis-hydroxyproline-treated cultures. However, accumulation of p65 in nerve fibers at sites of muscle contact, a sign of presynaptic differentiation, was unaffected by cis-hydroxyproline treatment. In addition, even in cis-hydroxyproline-inhibited cultures, agrin was evident at more than 90% of contacts showing accumulation of p65 in the nerve terminal. Therefore, a mechanism exists to maintain agrin concentrations at nerve-muscle contacts, even when at least some extracellular matrix (ECM) proteins are disrupted. Our results suggest that HSPG is not required for the induction of nerve terminal differentiation but are consistent with the idea that HSPG or other ECM proteins are important in both nerve- and agrin-induced AChR clustering. In particular, agrin accumulation at sites of nerve-muscle contact is not sufficient to induce AChR clusters when the ECM at these contacts is disrupted.

Agrin↗
PubMed1995

Patterns of presynaptic gene expression define two stages of synaptic differentiation.

To elucidate the sequence of molecular events leading to nerve terminal differentiation, we have examined the regulation of expression of presynaptic protein genes during synapse formation in vivo. In the chick ciliary ganglion (CG), synaptophysin IIa and synaptophysin IIb mRNAs showed threefold increases relative to neurofilament-M mRNA during the time of target contact [Embryonic Day 7 (E7)-E9]. Expression of synaptotagmin I mRNA also increased severalfold over this time interval. Thus, mRNAs for three synaptic vesicle proteins are upregulated coordinately during synaptogenesis. In contrast, the major increase in choline acetyltransferase (ChAT) mRNA (four- to fivefold) occurred between E15 and E20, coincident with the maturation of synapses in the CG. Coincident with ChAT upregulation, there is a switch in the relative abundance of mRNAs encoding vesicle protein isoforms. In particular, mRNAs encoding synaptophysin IIb and synaptotagmin II (which is undetectable at E9) become predominant. Therefore, although synaptic vesicle protein mRNAs are upregulated in a first phase of differentiation at the time of synapse formation, a temporally distinct phase of presynaptic protein gene regulation, associated with the specific maturation of synapses, is also apparent.

Amino Acid Sequence↗
PubMed1995

Agrin is a differentiation-inducing "stop signal" for motoneurons in vitro.

Proteins of the synaptic basal lamina are important in directing the differentiation of motor nerve terminals. One synaptic basal lamina protein, agrin, which influences postsynaptic muscle differentiation, has been suggested to influence nerve terminals as well. To test this hypothesis, we cocultured chick ciliary ganglion neurons with agrin-expressing CHO cells. Ciliary ganglion neurons, but not sensory neurons, adhered five times as well to agrin-expressing cells as to untransfected cells. Further, ciliary ganglion neurites were growth inhibited upon contact with agrin-expressing cells. Finally, the synaptic vesicle protein synaptotagmin became concentrated at contacts between ciliary ganglion neurites and agrin-expressing cells. These activities were shared by neuronal and muscle-derived agrin isoforms, consistent with the hypothesis that muscle agrin may influence the presynaptic axon. Our results suggest that agrin influences the growth and differentiation of motoneurons in vivo.

Agrin↗
PubMed1994

Ca2+ influx and neurite growth in response to purified N-cadherin and laminin.

The signaling mechanisms underlying neurite growth induced by cadherins and integrins are incompletely understood. In our experiments, we have examined these mechanisms using purified N-cadherin and laminin (LN). We find that unlike the neurite growth induced by fibroblastic cells expressing transfected N-cadherin (Doherty, P., and F.S. Walsh. 1992. Curr. Opin. Neurobiol. 2:595-601), growth induced by purified N-cadherin in chick ciliary ganglion (CG), sensory, or forebrain neurons is not sensitive to inhibition by pertussis toxin. Using fura-2 imaging of single cells, we show that soluble N-cadherin induces Ca2+ increases in CG neuron cell bodies, and, importantly, in growth cones. In contrast, N-cadherin can induce Ca2+ decreases in glial cells. N-cadherin-induced neuronal Ca2+ responses are sensitive to Ni2+, but are relatively insensitive to diltiazem and omega-conotoxin. Similarly, neurite growth induced by purified N-cadherin is inhibited by Ni2+, but is unaffected by diltiazem and conotoxin. Soluble LN also induced small Ca2+ responses in CG neurons. LN-induced neurite growth, like that induced by N-cadherin, is insensitive to diltiazem and conotoxin, but is highly sensitive to Ni2+ inhibition. K+ depolarization experiments suggest that voltage-dependent Ca2+ influx pathways in CG neurons (cell bodies and growth cones) are largely blocked by the combination of diltiazem and Ni2+. Our results demonstrate that cadherin signaling involves cell type-specific Ca2+ changes in responding cells, and in particular, that N-cadherin can cause Ca2+ increases in neuronal growth cones. Our findings are consistent with the current idea that distinct neuronal transduction pathways exist for cell adhesion molecules compared with integrins, but suggest that the involvement of Ca2+ signals in both of these pathways is more complex than previously appreciated.

Animals↗
PubMed1994

Cell adhesion molecules regulating neurite growth from amacrine and rod photoreceptor cells.

A great deal is now known about the cell adhesion molecules (CAMs) that are responsible for promoting the growth of ganglion cell axons as they project out of the retina through the optic nerve and finally to distant targets in the brain. However, the CAMs important for regulating axon outgrowth from nonprojection neurons, such as amacrine cells and rods, are not known. Such local circuit neurons extend their neurites rather short distances on cellular surfaces not normally encountered by the ganglion cell axons. To study the mechanisms regulating axon or dendrite growth from local circuit neurons, neurite outgrowth from amacrine cells and rod photoreceptor cells derived from the rat was examined in vitro on immunopurified forms of NCAM, L1, and N-cadherin, three well-characterized adhesive molecules found in the developing retina. Either early (P3) or late (P10) postnatal amacrine cells grew neurites on all three CAMs, but there were significant differences in the percentage of the amacrine cells that responded to each CAM. None of the CAMs supported neurite outgrowth from early postnatal rods, but, surprisingly, NCAM stimulated vigorous neurite extension from rods isolated at postnatal day 10. Postnatal ganglion cells were also examined for comparison and were found not to grow neurites on NCAM, but did grow extensive processes on L1 and N-cadherin. These results show that NCAM, L1, and N-cadherin can promote neurite outgrowth from local circuit neurons, but that the effectiveness of any particular CAM is dependent on the cell type and the developmental period.

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PubMed1993

Coordinate and noncoordinate regulation of synaptic vesicle protein genes during embryonic development.

Because the formation of a mature nerve terminal requires the accumulation of large quantities of synaptic vesicles, the expression of synaptic vesicle proteins would be expected to correlate with synaptogenesis. However, previous studies have provided conflicting evidence on this point. We have examined the developmental pattern of expression of mRNA and protein for three RNAs derived from two genes coding for synaptic vesicle membrane proteins. For these experiments, we cloned a chick p65 (synaptotagmin) cDNA using a reduced stringency screen with a rat p65 cDNA probe. We examined p65 expression in chick forebrain in conjunction with that of synaptophysin II. RNase protection assays for p65 and the two isoforms of synaptophysin II (Bixby, 1992) show essentially coordinate increases of these three mRNAs in embryonic forebrain during the peak period of synaptogenesis (E17 to E20). However, each of the three mRNAs has a distinct temporal pattern of expression during the early stages of embryogenesis. In the ciliary ganglion, upregulation of synaptophysin II mRNA correlates very well with synaptogenesis. Our results suggest that the regulation of expression of vesicle membrane protein mRNA can serve as a marker for synaptogenesis, despite temporal differences in early expression patterns. In contrast to mRNA expression, assays for vesicle protein expression show a relatively steady rise in both p65 and synaptophysin II throughout the embryonic period, without a sharp increase corresponding to that seen in message levels. These results suggest that the expression both of the p65 and of the synaptophysin II proteins is post-transcriptionally regulated.

Amino Acid Sequence↗
PubMed1993

Tyrosine phosphorylation in early embryonic growth cones.

A large and growing body of evidence suggests that the regulation of tyrosine phosphorylation is important in the induction of axon growth. We have examined the subcellular distribution of enzymes regulating tyrosine phosphorylation in early embryonic brain, employing a preparation of isolated growth cone particles (GCPs). Because of the early developmental age and well-characterized nature of our tissue source, our GCP preparation offers some advantages over those described previously. As was found with other GCPs, our GCPs had relatively high levels of both the growth-associated protein GAP-43 and the intracellular tyrosine kinase pp60c-arc. In addition, we found that both total tyrosine kinase activity and total tyrosine phosphatase activity were concentrated two- to threefold in the GCPs relative to a neuronal membrane fraction. Two other nonreceptor tyrosine kinases, YES and FYN, were concentrated in the GCPs to a similar degree as that seen for SRC. In addition, we examined the developmental expression in brain of the three tyrosine kinases, using both a quantitative ELISA and Western blot analysis. Our results show that FYN, like SRC, reaches a peak of expression early in development, and declines thereafter. In contrast, expression of YES peaks later, and remains high in the adult brain. Immunofluorescence staining suggests that FYN is expressed both by neurons and by glia, and possibly by neuronal precursor cells. Our results implicate multiple tyrosine kinases as well as tyrosine phosphatases in growth cone function. In addition, the concentration of FYN in early embryonic growth cones combined with its early peak of expression suggests an important role for FYN in early neuronal development.

Amino Acid Sequence↗
PubMed1992

Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth.

Protein tyrosine kinases (PTKs) have major roles in signal transduction and growth control. There are several lines of evidence implicating PTKs in the regulation of axon growth, and this has led to the suggestion that they are centrally involved in the transduction of neuronal growth signals. To test this idea, we assayed the effect of the compounds genistein and lavendustin, specific inhibitors of PTKs, on neurite growth. We find that genistein greatly reduces phosphotyrosine in neurons, as expected from its action on other cells. Surprisingly, administration of genistein or lavendustin potentiated substrate-induced neurite growth in at least several different neuronal types. Stimulation of neurite growth by genistein was abolished by vanadate, providing additional evidence that inhibition of PTKs is responsible for this effect. The potentiation of growth is rather general, in that it occurs on several different extracellular matrix substrates and on two different cell adhesion molecules. Both the initiation of neurite growth and the rate of neurite elongation appear to be potentiated. Our results do not provide evidence for models of substrate-induced signal transduction that involve PTKs as a positive and necessary step, but suggest that such kinases play a regulatory role in neurite elongation.

Animals↗
PubMed1992

Identification of an alternatively spliced avian member of the synaptophysin gene family.

Synaptic vesicle membrane proteins are important in the release of neurotransmitters and as markers of presynaptic differentiation in neurons, and the synaptophysins are a major class of synaptic vesicle proteins. By low stringency screening of a chick brain cDNA library with a rat synaptophysin probe, we have isolated cDNAs that encode a novel member of the synaptophysin/synaptoporin family. Two different protein-coding forms of the cDNA were found, apparently generated through alternative splicing of a single gene. The deduced proteins, called synaptophysin IIa and synaptophysin IIb, share 258 amino acids (starting from position 10 in IIa and position 30 in IIb), that are most closely related to the rat synaptoporin sequence. The N-terminal sequence of IIa is similar to that of rat synaptoporin, and the N-terminal sequence of IIb is similar to that of rat synaptophysin. Northern blot analysis and nuclease protection experiments demonstrate that IIa and IIb are expressed in a variety of brain regions, the spinal cord, and dorsal root ganglia, but not in non-neuronal tissues. Further, the two splice variants are differentially distributed. In most brain regions the IIb form predominates, and the cerebellum appears to express only the IIb form, but the IIa form is relatively elevated in peripheral neurons. Western blot analysis with an antibody to a synthetic peptide common to both forms demonstrates the expression of synaptophysin II as a 39 kDa protein, apparently distinct from synaptophysin (40 kDa). The results suggest that the regulation and function of the synaptophysin gene family is more complex than had been appreciated.

Amino Acid Sequence↗
PubMed1990

Purified N-cadherin is a potent substrate for the rapid induction of neurite outgrowth.

N-cadherin is the predominant mediator of calcium-dependent adhesion in the nervous system (Takeichi, M. 1988. Development (Camb.). 102: 639-655). Investigations using antibodies to block N-cadherin function (Bixby, J.L., R.L. Pratt, J. Lilien, and L.F. Reichardt. 1987. Proc. Natl. Acad. Sci. USA. 84:2555-2569; Bixby, J.L., J. Lilien, and L.F. Reichardt. 1988. J. Cell Biol. 107:353-362; Tomaselli, K.J., K.N. Neugebauer, J.L. Bixby, J. Lilien, and L.F. Reichardt. 1988. Neuron. 1:33-43) or transfection of the N-cadherin gene into heterologous cell lines (Matsunaga, M., K. Hatta, A. Nagafuchi, and M. Takeichi. 1988. Nature (Lond.). 334:62-64) have provided evidence that N-cadherin, alone or in combination with other molecules, can participate in the induction of neurite extension. We have developed an affinity purification procedure for the isolation of whole N-cadherin from chick brain and have used the isolated protein as a substrate for neurite outgrowth. N-cadherin promotes the rapid extension of neurites from chick ciliary ganglion neurons, which extend few or no neurites on adhesive but noninducing substrates such as polylysine, tissue culture plastic, and collagens. N-cadherin is extremely potent, more so than the L1 adhesion molecule, and comparable to the extracellular matrix protein laminin. Compared to laminin, however. N-cadherin promotes outgrowth from ciliary ganglion neurons extremely rapidly and with a distinct morphology. These results provide a direct demonstration that N-cadherin is sufficient to induce neurite outgrowth when substrate bound and suggest that the mechanism(s) involved may differ from that induced by laminin.

Animals↗
PubMed1990

Extracellular matrix molecules and cell adhesion molecules induce neurites through different mechanisms.

It has recently become clear that both extracellular matrix (ECM) glycoproteins and various cell adhesion molecules (CAMs) can promote neurite outgrowth from primary neurons, though little is known of the intracellular mechanisms through which these signals are transduced. We have previously obtained evidence that protein kinase C function is an important part of the neuronal response to laminin (Bixby, J.L. 1989. Neuron. 3:287-297). Because such CAMs as L1 (Lagenauer, C., and V. Lemmon. 1987. Proc. Natl. Acad. Sci. USA. 84:7753-7757) and N-cadherin (Bixby, J.L. and R. Zhang. 1990. J. Cell Biol. 110:1253-1260) can be purified and used as substrates to promote neurite growth, we have now tested whether the response to CAMs is similarly dependent on protein kinase C. We find that inhibition of protein kinase C inhibits growth on fibronectin or collagen as well as on laminin. In contrast, C kinase inhibition actually potentiates the initial growth response to L1 or N-cadherin. The later "phase" of outgrowth on both of these CAMs is inhibited, however. Additionally, phorbol esters, which have no effect on neurite growth when optimal laminin concentrations are used, potentiate growth even on optimal concentrations of L1 or N-cadherin. The results indicate that different intracellular mechanisms operate during initial process outgrowth on ECM substrates as compared to CAM substrates, and suggest that protein kinase C function is required for continued neurite growth on each of these glycoproteins.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Protein kinase C is involved in laminin stimulation of neurite outgrowth.

We are investigating the intracellular events involved in the induction of neurite outgrowth. The phorbol ester TPA, an activator of protein kinase C, potentiates neurite outgrowth from ciliary ganglion neurons cultured on suboptimal laminin concentrations, but not on optimal laminin concentrations. TPA also stimulates growth on fibronectin and collagen similar to that observed on laminin under control conditions. Manipulations that elevate intracellular cAMP levels (expected to activate A kinase) reduce neurite outgrowth on laminin. The protein kinase C inhibitors H7 and sphingosine inhibit neurite outgrowth on laminin in a reversible and dose-dependent manner. H7 does not inhibit the process outgrowth induced by concanavalin A in the same neurons. The results suggest that activation of protein kinase C is an important step in the neurite outgrowth caused by laminin binding to its receptor(s).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

N-cadherin and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces.

Receptor-mediated interactions between neurons and astroglia are likely to play a crucial role in the growth and guidance of CNS axons. Using antibodies to neuronal cell surface proteins, we identified two receptor systems mediating neurite outgrowth on cultured astrocytes. N-cadherin, a Ca2(+)-dependent cell adhesion molecule, functions prominently in the outgrowth of neurites on astrocytes by E8 and E14 chick ciliary ganglion (CG) neurons. beta 1-class integrin ECM receptor heterodimers function less prominently in E8 and not at all in E14 neurite outgrowth on astrocytes. The lack of effect of integrin beta 1 antibodies on E14 neurite outgrowth reflects an apparent loss of integrin function, as assayed by E14 neuronal attachment and process outgrowth on laminin. N-CAM appeared not to be required for neurite outgrowth by either E8 or E14 neurons. Since N-cadherin and integrin beta 1 antibodies together virtually eliminated E8 CG neurite outgrowth on cultured astrocytes, these two neuronal receptors are probably important in regulating axon growth on astroglia in vivo.

Animals↗
PubMed1988

Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro.

Schwann cells have a unique role in regulating the growth of axons during regeneration and presumably during development. Here we show that Schwann cells are the best substrate yet identified for promoting process growth in vitro by peripheral motor neurons. To determine the molecular interactions responsible for Schwann cell regulation of axon growth, we have examined the effects of specific antibodies on process growth in vitro, and have identified three glycoproteins that play major roles. These are the Ca2+-independent cell adhesion molecule (CAM), L1/Ng-CAM; the Ca2+-dependent CAM, N-cadherin; and members of the integrin extracellular matrix receptor superfamily. Two other CAMs present on neurons and/or Schwann cells-N-CAM and myelin-associated glycoprotein-do not appear to be important in regulating process growth. Our results imply that neuronal growth cones use integrin-class extracellular matrix receptors and at least two CAMs--N-cadherin and L1/Ng-CAM-for growth on Schwann cells in vitro and establish each of these glycoproteins as a strong candidate for regulating axon growth and guidance in vivo.

Animals↗