We have previously demonstrated that the growth of peripheral nervous system axons is strongly attracted towards limb buds and skin explants in vitro. Here, we show that directed axonal growth towards skin explants of Xenopus laevis in matrigel is associated with expression of matrix metalloproteinase (MMP)-18 and also other MMPs, and that this long-range neurotropic activity is inhibited by the broad-spectrum MMP inhibitors BB-94 and GM6001. We also show that forced expression of MMP-18 in COS-7 cell aggregates enhances axonal growth from Xenopus dorsal root ganglia explants.
View Article and Find Full Text PDFThe molecular basis of axonal regeneration of central nervous system (CNS) neurons remains to be fully elucidated. In part, this is due to the difficulty in maintaining CNS neurons in vitro. Here, we show that dissociated neurons from the cerebral cortex and hippocampus of adult mice may be maintained in culture for up to 9 days in defined medium without added growth factors.
View Article and Find Full Text PDFLaminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, β2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm-Swarm tumor (EHS-laminin) but its T(m) value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the β chain reduced the stability of the coiled-coil structure of laminin-121.
View Article and Find Full Text PDFAxonal regeneration is enhanced by the prior ;conditioning' of peripheral nerve lesions. Here we show that Xenopus dorsal root ganglia (DRG) with attached peripheral nerves (PN-DRG) can be conditioned in vitro, thereafter showing enhanced neurotrophin-induced axonal growth similar to preparations conditioned by axotomy in vivo. Actinomycin D inhibits axonal outgrowth from freshly dissected PN-DRG, but not from conditioned preparations.
View Article and Find Full Text PDFThe subpopulation of dorsal root ganglion (DRG) neurons recognized by Griffonia simplicifolia isolectin B4 (IB4) differ from other neurons by expressing receptors for glial cell line-derived neurotrophic factor (GDNF) rather than neurotrophins. Additionally, IB4-labeled neurons do not express the laminin receptor, alpha7-integrin (Gardiner et al., 2005), necessary for optimal axonal regeneration in the peripheral nervous system.
View Article and Find Full Text PDFNon-viral methods of transfection of cDNAs into adult neurons and other post-mitotic cells are generally very inefficient. However, the recent development of Nucleofector technology developed by Amaxa Biosystems allows direct delivery of cDNAs into the nucleus, enabling transfection of non-dividing cells. In this study, we describe a reliable method for culturing large numbers of retinal cells from adult rats and using Nucleofection, we were able to transfect cDNA-encoding GFP (jellyfish green fluorescent protein) into retinal ganglion cells (RGCs) with relatively high efficiency (up to 28%).
View Article and Find Full Text PDFThe mechanisms for directing axons to their targets in developing limbs remain largely unknown though recent studies in mice have demonstrated the importance of neurotrophins in this process. We now report that in co-cultures of larval Xenopus laevis limb buds with spinal cords and dorsal root ganglia of Xenopus and axolotl (Ambystoma mexicanum) axons grow directly to the limb buds over distances of up to 800 microm and in particular to sheets of epidermal cells which migrate away from the limb buds and also tail segments in culture. This directed axonal growth persists in the presence of trk-IgG chimeras, which sequester neurotrophins, and k252a, which blocks their actions mediated via trk receptors.
View Article and Find Full Text PDFConditioning lesions of peripheral nerves improve axonal regeneration after injury and involve changes in expression of proteins required for axonal growth. Integrin alpha7beta1 expression in motor and sensory neurons increases following nerve lesions and motor axon regeneration is impaired in alpha7 integrin KO mice (J. Neurosci.
View Article and Find Full Text PDFMol Cell Neurosci
October 2002
Recent studies have shown that lens lesion promotes axonal regeneration in the optic nerve of adult rats. In the present investigations, dissociated retinal ganglion cells (RGC) from intact postnatal (P) 9-11 rats showed spontaneous neurite outgrowth on laminin-1, in contrast to RGC from intact P14-adult rats. Neurite outgrowth from P9-14 RGC on laminin-1 was promoted by prior lens lesion and also during coculture with lesioned lenses.
View Article and Find Full Text PDFBradykinin B2 receptor mRNA was detected at low levels, both by RT-PCR and by in situ hybridization, in freshly isolated dorsal root ganglia (DRG) and in ganglia cultured in the absence of neurotrophic factors, but was strongly upregulated by culture in the presence of nerve growth factor (NGF). The effect of NGF is mediated via TrkA receptors. The related neurotrophins, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4, were ineffective in upregulating B2 mRNA, but a small upregulation was seen with the unrelated neurotrophin glial cell line-derived neurotrophic factor (GDNF).
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