α7 integrin mediates neurite outgrowth of distinct populations of adult sensory neurons

https://doi.org/10.1016/j.mcn.2004.08.017Get rights and content

Abstract

The successful regeneration of peripheral branches of sensory neurons following injury is attributed to the presence of neurotrophins and interaction of regenerating axons with the extracellular matrix. Here, we show that the laminin receptor, α7β1 integrin is a crucial mediator of neurite outgrowth from distinct populations of sensory neurons. Following sciatic nerve crush, α7 integrin is expressed by medium–large diameter, NF200-immunoreactive (IR), and medium diameter, CGRP-IR, neurons, but very few small diameter non-peptidergic neurons. The functional significance of α7 integrin expression following injury was addressed using dissociated adult rat and mouse sensory neurons. By using function-blocking antibodies and neurons isolated from α7 integrin null mice, we demonstrate that NGF- and NT-3-stimulated neurite outgrowth is reduced in the absence of α7 integrin signaling. In contrast, GDNF-stimulated neurite outgrowth is less dependent on α7 integrin. These results define an essential interaction between α7 integrin and laminin for mediating neurite outgrowth of subpopulations of injured adult sensory neurons.

Introduction

The successful regeneration of peripheral branches of sensory neurons following injury is largely attributed to the presence of neurotrophins and a permissive growth substratum rich in extracellular matrix (ECM) components that promote axonal regeneration (Fu and Gordon, 1997, Ide, 1996). This has been utilised in the design of nerve conduits, made from a variety of biological or non-biological materials, to provide both mechanical support and guidance for regenerating axons following nerve injury. These conduits may be filled with ECM components which support growth and further manipulated to provide an environment optimised for axonal regeneration. For example, the addition of neurotrophins or cytokines into the conduit enhances regeneration (McKay Hart et al., 1997, Sterne et al., 1997, Whitworth et al., 1995).

Integrins are heterodimeric ECM receptors which are expressed at high levels in the developing nervous system and are present in the central domain and leading edge of growth cones. They are important in mediating axonal guidance, adhesion and migration (Letourneau and Shattuck, 1989, Renaudin et al., 1999, Tomaselli et al., 1993). Many neuronal integrins are regulated postnatally (Guan et al., 2003, Hammarberg et al., 2000) and an age-related decrease in integrin levels has been correlated with reduced neurite outgrowth in both retinal (Cohen et al., 1986) and sensory neurons (Condic, 2001). The ECM plays a vital role in mediating axonal regeneration following injury in the adult nervous system. Following nerve injury, there is a rapid upregulation of many ECM proteins, including fibronectin (Lefcort et al., 1992, Vogelezang et al., 1999) and laminin (Wallquist et al., 2002). Experiments utilising function-blocking antibodies have highlighted the essential role laminin plays in promoting axonal regeneration (Agius and Cochard, 1988, Ide, 1996).

Adult sensory neurons are a heterogenous population which may be divided into subpopulations that differ in neurochemistry, neuroanatomy, trophic requirements and sensory modality (Lawson, 1995, McMahon et al., 1994). The neuronal response to injury is correspondingly diverse, and the mechanisms that control survival and regenerative programmes are poorly understood. To date, five laminin-binding integrins have been identified in sensory neurons: α1β1, α3β1, α6β1, α6β4 and α7β1 (Tomaselli et al., 1993, Werner et al., 2000) and studies using function-blocking antibodies have revealed the β1-associated integrins to be important in mediating adhesion and neurite outgrowth (Agius and Cochard, 1988, Condic and Letourneau, 1997, Ekstrom et al., 2003, Tomaselli et al., 1993). Less, however, is known about the α integrin subunits that are involved in axon regeneration. Since different populations of sensory neurons express different integrin subunits during embryonic development (Guan et al., 2003), we hypothesise that the same might be true in the adult sensory nervous system following injury. Successful regeneration may be mediated by the discrete spatial and temporal distribution of integrins, governing the responsiveness of neurons to specific ECM components.

In this study, we perform a detailed analysis of α7 integrin expression in rat dorsal root ganglia (DRG) and sciatic nerve at various time points following a sciatic nerve crush. Our data indicate a dynamic upregulation of α7 integrin in specific populations of sensory neurons. We utilise function-blocking antibodies and genetically altered mice to address the role of α7 integrin. Importantly, we demonstrate a requirement for α7 integrin in mediating neurite outgrowth that is restricted to distinct subpopulations of sensory neurons.

Section snippets

Sensory nerve regeneration is impaired in α7 integrin −/− mice

In order to determine whether α7 integrin is necessary for successful regeneration of injured sensory neurons, we examined growth-associated protein (GAP)-43-immunoreactivity (IR), a commonly used marker for regenerating axons, in the sciatic nerve of wild-type and α7 integrin −/− mice 2 days following nerve crush (Fig. 1). We found that regeneration is impaired in the α7 integrin −/− mice (Figs. 1B, C) compared to wild-type mice (Figs. 1A, C). The distance migrated by GAP43-IR axons (Figs. 1A,

Discussion

It has long been known that integrins have a critical role in nerve regeneration via their ability to drive directed axon growth over ECM. However, the specific α heterodimers that are involved with axon regeneration in adult sensory neurons have not been fully elucidated. It now emerges from our work that α7β1 integrin has a critical role. We describe a dynamic upregulation of α7 integrin in distinct populations of neurons following peripheral nerve injury and show that α7β1 integrin is

Adult sensory neuron culture

Adult male and female α7 integrin −/− mice obtained from heterozygous crossings on a 129 Sv background (Mayer et al., 1997), adult male C57BL6 mice (30 g, Charles River, UK) and adult male Wistar rats (250 g, Charles River, UK) were used in this study. Animals were killed by concussion followed by decapitation. DRG were removed, cleaned of connective tissue and chemically dissociated in 0.125% collagenase in Ham's F12 (Gibco) for 1.5 h (Cafferty et al., 2001, Gavazzi et al., 1999). DRG were

Acknowledgment

This work was supported by a BBSRC Neuron Initiative award.

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    Current address: Department of Pharmacology and Therapeutics, St. Boniface Research Centre, R4046-351 Tache, Winnipig, MB, Canada, R2H 2A6.

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