Immunocytochemical localization of a cell adhesion molecule, integrin α5β1, in nerve growth cones

doi:10.1007/s007760050116

Journal of Orthopaedic Science

Volume 4, Issue 5, September 1999, Pages 353-360

https://doi.org/10.1007/s007760050116 Get rights and content

Abstract

It is well-known that nerve growth cones, the growing tips of nerves, play a central role in determining the direction taken by regenerating peripheral nerves though neurotropism and contact guidance. In order to identify the molecules expressed on growth cones that are responsible for contact guidance, we investigated the possible involvement of integrins as sensors for the extracellular matrix. In cultured rat PC-12i cells and chick dorsal root ganglion cells, we found that integrin α5β1 was concentrated in the filopodia and central regions of the growth cones. These integrin α5β1-rich regions corresponded well with the sites where the growth cones came into contact with and adhered to the extracellular matrix. Integrin α5β1 has been reported to bind with fibronectin in the extracellular matrix. When examined by triple staining and confocal laser scanning microscopy, we found that the distribution of integrin α5β1 in the growth cones was very similar to that of actin filaments. Integrin α5β1 was also expressed by Schwann cells. On immuno-electron microscopy, integrin α5β1 was also identified in regenerating axons in vivo. These results suggest that integrin α5β1 expressed on growth cones may function as a sensor for the extracellular matrix and Schwann cells, and thus mediate functionally important interactions in the development and regeneration of peripheral nerves.

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An integrin-binding array platform identifies α<inf>v</inf>β<inf>8</inf> and α<inf>5</inf>β<inf>1</inf> integrins on rat primary cortical neurons to support their survival and growth
2020, Journal of Neuroscience Methods
Citation Excerpt :
It binds to the isoforms of fibronectin and osteopontin (Humphries, Byron et al. 2006), and is necessary for neurite outgrowth of some specific neurons such as dopaminergic neurons (Izumi, Wakita et al. 2017). The α5β1 integrin is also involved in regeneration of peripheral nerve following injury as increased expression was observed in axon growth cones (Yanagida, Tanaka et al. 1999). The α3β1 integrin has been known as a major receptor to laminin (Nishiuchi, Murayama et al. 2003), the major type of ECM proteins in the brain.
Integrins are the major cell adhesion receptors expressed in almost all cell types connecting the extracellular matrix with cell cytoskeletons and transducing bi-directional signals across cell membranes. In the central nervous system (CNS), integrins are pivotal for CNS cell migration, differentiation, neurite outgrowth and synaptogenesis in both physiological and pathological conditions. Here we studied the effect of different integrin biding peptides for growth and development of primary cortical neurons in vitro.
Rat primary cortical neurons were cultured in an integrin-binding array platform, which contains immobilized varying short synthetic peptides that bind to 16 individual types of integrin on a 48-well cell culture plate. After cultured for 7 days, cells were fixed and processed for immunostaining with neuronal markers. The overall neuronal growth and neurite outgrowths were quantified.
We found that binding peptides for integrin α_vβ₈, α₅β₁ and α₃β₁ particularly the former two provided superior condition for neuronal growth, survival and maturation. Moreover, optimal neurite outgrowth was observed when neurons were cultured in 3-dimension using injectable hydrogel along with binding peptide for α_vβ₈ or α₅β₁ integrins.
For primary neuronal culture, poly-D-lysine coating is conventional method to support cell attachment. Our study has demonstrated that selected integrin binding peptides provide greater support for the growth of cultured primary neurons.
These data suggest that integrin α_vβ₈ and α₅β₁ are conducive for survival, growth and maturation of primary cortical neurons. This information could be utilized in designing combinational biomaterial and cell-based therapy for neural regeneration following brain injury.
Fibronectin supports neurite outgrowth and axonal regeneration of adult brain neurons in vitro
2012, Brain Research
Citation Excerpt :
FN is known to act as a ligand for at least 11 different integrin heterodimers (Leiss et al., 2008) supporting adhesion of numerous cell types and also neurite outgrowth from developing peripheral nervous system (PNS) and central nervous system (CNS) neurons (Akers et al., 1981; Carri et al., 1988; Haugen et al., 1992; Humphries et al., 1988; Rogers et al., 1985; 1988). In adult animals, FN is up-regulated in lesioned peripheral nerves (Lefcort et al., 1992; Siironen et al., 1992) and α5β1 integrin, an important FN receptor, is expressed on regenerating axons (Lefcort et al., 1992; Yanagida et al., 1999) and mediates neurite outgrowth from dorsal root ganglion (DRG) neurons on FN in vitro (Gardiner et al., 2007). In the CNS, α5β1 integrin is expressed by neurons during development and although down-regulated during maturation (Yoshida et al., 2003) is still detectable in the brains of mature animals (King et al., 2001) including neurons in the hippocampus and cerebral cortex (Bi et al., 2001).
The 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. Outgrowth of neurites including axons was observed from both CNS sources and was significantly greater on plasma fibronectin than on other substrata such as laminin and merosin. Neurite outgrowth on fibronectin appears to be mediated by α5β1 integrin since a recombinant fibronectin fragment containing binding sites for this receptor was as effective as intact fibronectin in supporting neurite outgrowth. Conversely, function-blocking antibodies to α5 and β1 integrin sub-units inhibited neurite outgrowth on intact fibronectin. These results suggest that the axonal regeneration seen in in vivo studies using fibronectin-based matrices is due to the molecule itself and not a consequence of secondary events such as cellular infiltration. They also indicate the domains of fibronectin that may be responsible for eliciting this response.
Integrins as receptor targets for neurological disorders
2012, Pharmacology and Therapeutics
Citation Excerpt :
The β1-integrin receptors were first identified as mediators of neurite outgrowth on ECM components (Reichardt & Tomaselli, 1991). Since then at least 14 of the 24 known integrin subunit combinations have been reported in nervous system: α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α8β1, α9β1, αvβ1, αvβ3, αvβ6, αvβ8, and α6β4 (Table 1 and Fig. 2) (Pinkstaff et al., 1999; Renaudin et al., 1999; Yanagida et al., 1999; Chun et al., 2001; Chan et al., 2003; Morini & Becchetti, 2010). Different heterodimers of integrin have different affinities to the various ECM ligands and will be activated according to ligand and generate a transient or persistent signals.
This review focuses on the neurobiology of integrins, pathophysiological roles of integrins in neuroplasticity and nervous system disorders, and therapeutic implications of integrins as potential drug targets and possible delivery pathways. Neuroplasticity is a central phenomenon in many neurological conditions such as seizures, trauma, and traumatic brain injury. During the course of many brain diseases, in addition to intracellular compartment changes, alterations in non-cell compartments such as extracellular matrix (ECM) are recognized as an essential process in forming and reorganizing neural connections. Integrins are heterodimeric transmembrane receptors that mediate cell–ECM and cell–cell adhesion events. Although the mechanisms of neuroplasticity remain unclear, it has been suggested that integrins undergo plasticity including clustering through interactions with ECM proteins, modulating ion channels, intracellular Ca²⁺ and protein kinase signaling, and reorganization of cytoskeletal filaments. As cell surface receptors, integrins are central to the pathophysiology of many brain diseases, such as epilepsy, and are potential targets for the development of new drugs for neurological disorders.
Integrin signaling is integral to regeneration
2008, Experimental Neurology
The inability of the adult injured mammalian spinal cord to successfully regenerate is not well understood. Studies suggest that both extrinsic and intrinsic factors contribute to regeneration failure. In this review, we focus on intrinsic factors that impact regeneration, in particular integrin receptors and their downstream signaling pathways. We discuss studies that address the impact of integrins and integrin signaling pathways on growth cone guidance and motility and how lessons learned from these studies apply to spinal cord regeneration in vivo.
Preconditioning injury-induced neurite outgrowth of adult rat sensory neurons on fibronectin is mediated by mobilisation of axonal α5 integrin
2007, Molecular and Cellular Neuroscience
A preconditioning sciatic nerve crush promotes the capacity of adult sensory neurons to regenerate following a subsequent injury to their axons. The increase in regeneration is detected in cultures of dissociated neurons, as an earlier and enhanced rate of neurite elongation.
We compare neurotrophin-stimulated neurite outgrowth from sensory neurons on laminin and fibronectin. There is a poor response of sensory neurons to fibronectin in comparison to laminin, but this is enhanced by a preconditioning lesion to the sciatic nerve 7 days prior to culture. By using specific integrin-binding fibronectin fragments and function-blocking antibodies, we demonstrate that the enhanced preconditioned neurite outgrowth on fibronectin is largely mediated by α5β1 integrin. Preconditioning injury alter the subcellular localisation of α5 integrin in preconditioned neurites. We show that α5 integrin localises to adhesion complexes in the growth cone and neurites of preconditioned neurons, but not control neurons.
Mats made from fibronectin support oriented growth of axons in the damaged spinal cord of the adult rat
2003, Experimental Neurology
A variety of biological as well as synthetic implants have been used to attempt to promote regeneration into the damaged spinal cord. We have implanted mats made from fibronectin (FN) into the damaged spinal cord to determine their effectiveness as a substrate for regeneration of axons. These mats contain oriented pores and can take up and release growth factors. Lesion cavities 1 mm in width and depth and 2 mm in length were created on one side of the spinal cord of adult rats. FN mats containing neurotrophins or saline were placed into the lesion. Mats were well integrated into surrounding tissue and showed robust well-oriented growth of calcitonin gene-related peptide, substance P, GABAergic, cholinergic, glutamatergic, and noradrenergic axons into FN mats. Transganglionic tracing using cholera toxin B indicated large-diameter primary afferents had grown into FN implants. Schwann cells had also infiltrated FN mats. Electron microscopy confirmed the presence of axons within implants sites, with most axons either ensheathed or myelinated by Schwann cells. Mats incubated in brain-derived neurotrophic factor and neurotrophin-3 showed significantly more neurofilament-positive and glutamatergic fibers compared to saline- and nerve growth factor-incubated mats, while mats incubated with nerve growth factor showed more calcitonin gene-related peptide-positive axons. In contrast, neurotrophin treatment had no effect on PGP 9.5-positive axons. In addition, in some animals with neurotrophin-3-incubated mats, cholera toxin B-labelled fibers had grown from the mat into adjoining intact areas of spinal cord. The results indicate that FN mats provide a substrate that is permissive for robust oriented axonal growth in the damaged spinal cord, and that this growth is supported by Schwann cells.

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Journal of Orthopaedic Science

Abstract

References (21)

In vitro guidance of retinal ganglion cell axons by RAGS, a 25-kDa tectal protein related to ligands for Eph receptor tyrosine kinases

Cell

Integrins: a family of cell surface receptors

Cell

Integrins: versatility, modulation, and signaling in cell adhesion

Cell

Schwann cell basal lamina and nerve regeneration

Brain Res

Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord

Cell

Localization of Rabphilin-3A on the synaptic vesicle

Biochem Biophys Res Commun

Synaptophysin immunocytochemistry in the regenerating sprouts from the nodes of Ranvier in injured rat sciatic nerve

Brain Res

The netrins define a family of axon outgrowth-promoting proteins homologous to Celegans UNC-6

Cell

Transmembrane molecular assemblies in cell-extracellular matrix interactions

Curr Opin Cell Biol

Degeneration and regeneration of the nervous system

Cited by (24)

An integrin-binding array platform identifies α<inf>v</inf>β<inf>8</inf> and α<inf>5</inf>β<inf>1</inf> integrins on rat primary cortical neurons to support their survival and growth

Fibronectin supports neurite outgrowth and axonal regeneration of adult brain neurons in vitro

Integrins as receptor targets for neurological disorders

Integrin signaling is integral to regeneration

Preconditioning injury-induced neurite outgrowth of adult rat sensory neurons on fibronectin is mediated by mobilisation of axonal α5 integrin

Mats made from fibronectin support oriented growth of axons in the damaged spinal cord of the adult rat

Original articlesImmunocytochemical localization of a cell adhesion molecule, integrin α5β1, in nerve growth cones

Abstract

Cell

Cell

Cell

Brain Res

Cell

Biochem Biophys Res Commun

Brain Res

Cell

Curr Opin Cell Biol

Degeneration and regeneration of the nervous system

Original articles
Immunocytochemical localization of a cell adhesion molecule, integrin α5β1, in nerve growth cones