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Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with alpha4beta1 integrins.

ABSTRACT: The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for alpha4beta1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the alpha4 integrin cytoplasmic domain in neurite outgrowth.Expression of alpha4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type alpha4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal alpha4-mediated migration in leucocytes requires spatial regulation of alpha4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this alpha4(S988D), which mimics phosphorylated alpha4, did not promote neurite outgrowth. However, alpha4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable alpha4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin.We establish the importance of the alpha4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of alpha4 for extension of neuronal growth cones and migration of non-neural cells.

SUBMITTER: Vogelezang M

PROVIDER: S-EPMC1940015 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with alpha4beta1 integrins.

Vogelezang Mariette M Forster Ulrike B UB Han Jaewon J Ginsberg Mark H MH ffrench-Constant Charles C

BMC neuroscience 20070629

<h4>Background</h4>The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for alpha4beta1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the alpha4 integrin cytoplasmic domain in neurite outgrowth.<h4>Results</h4>Expression of alpha4 with mutations in the paxillin-binding domain re ...[more]

PMID: 17603879

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Project description:We have undertaken a genome-wide study of transcriptional activity in embryonic superior cervical ganglia (SCG) and dorsal root ganglia (DRG) during a time course of neurite outgrowth in vitro. Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result of tissue dissection. Comparison across both models revealed many genes with similar gene expression patterns during neurite outgrowth. These patterns were minimally affected by exposure to the potent inhibitory cue Semaphorin3A, indicating that this extrinsic cue does not exert major effects at the level of nuclear transcription. We also compared our data to several published studies of DRG and SCG gene expression in animal models of regeneration, and found the expression of a large number of genes in common between neurite outgrowth in vitro and regeneration in vivo. Experiment Overall Design: We wished to determine the transcriptional profiles of neurons undergoing neurite outgrowth in vitro. We were particularly interested in finding genes whose expression is generally associated with the process of neurite outgrowth, rather than with cell type-specific effects. Thus, in order to avoid focusing on transcripts unique to one tissue type versus another, we used a comparative strategy to look for effects that were common to two tissue types and therefore more likely to be involved in the general process of neurite outgrowth. While these explants contain multiple cell types, we felt this was preferable to the more disruptive conditions required to dissociate neurons or obtain a pure neuron population. To this end, we monitored gene expression in cultured explants from SCG and DRG using DNA microarrays. We initiated our studies by culturing embryonic day 13 (E13) mouse SCG in vitro and harvesting tissue for RNA isolation at time points from 2 to 65 hours. Time points were selected to detect both fast, short-term responses (2, 5 and 12 hours), as well as sustained, long-term changes (24, 40, and 65 hours). Samples were hybridized to Affymetrix MG-U74v2 A and B microarrays, with RNA from acutely dissected explants serving as a baseline reference. We followed these experiments with a parallel analysis of a more heterogeneous tissue type, the DRG, which is more frequently used than SCG for in vivo studies of neurite regeneration. Cervical and upper thoracic DRG from E12 embryos were cultured with NGF (the same trophic support as in SCG cultures), harvested at time points from 2 to 40 hours, and hybridized to Affymetrix MOE 430A microarrays.

Project description:BackgroundThe ability of a neuron to regenerate functional connections after injury is influenced by both its intrinsic state and also by extrinsic cues in its surroundings. Investigations of the transcriptional changes undergone by neurons during in vivo models of injury and regeneration have revealed many transcripts associated with these processes. Because of the complex milieu of interactions in vivo, these results include not only expression changes directly related to regenerative outgrowth and but also unrelated responses to surrounding cells and signals. In vitro models of neurite outgrowth provide a means to study the intrinsic transcriptional patterns of neurite outgrowth in the absence of extensive extrinsic cues from nearby cells and tissues.ResultsWe have undertaken a genome-wide study of transcriptional activity in embryonic superior cervical ganglia (SCG) and dorsal root ganglia (DRG) during a time course of neurite outgrowth in vitro. Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result of tissue dissection. Comparison across both models revealed many genes with similar gene expression patterns during neurite outgrowth. These patterns were minimally affected by exposure to the potent inhibitory cue Semaphorin3A, indicating that this extrinsic cue does not exert major effects at the level of nuclear transcription. We also compared our data to several published studies of DRG and SCG gene expression in animal models of regeneration, and found the expression of a large number of genes in common between neurite outgrowth in vitro and regeneration in vivo.ConclusionMany gene expression changes undergone by SCG and DRG during in vitro outgrowth are shared between these two tissue types and in common with in vivo regeneration models. This suggests that the genes identified in this in vitro study may represent new candidates worthy of further study for potential roles in the therapeutic regrowth of neuronal connections.

Dataset Information

Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with alpha4beta1 integrins.

Publications

Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with alpha4beta1 integrins.

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