Elsevier

Methods in Enzymology

Volume 426, 2007, Pages 203-221
Methods in Enzymology

Studies on Integrins in the Nervous System

https://doi.org/10.1016/S0076-6879(07)26010-0Get rights and content

Abstract

Integrins are of interest to neuroscientists because they and many of their ligands are widely expressed in the nervous system and have been shown to have diverse roles in neural development and function (Clegg et al., 2003; Li and Pleasure, 2005; Pinkstaff et al., 1998, 1999; Reichardt and Tomaselli, 1991; Schmid et al., 2005). Integrins have also been implicated in control of pathogenesis in several neurodegenerative diseases, brain tumor pathogenesis, and the aftermath of brain and peripheral nervous system injury (Condic, 2001; Ekstrom et al., 2003; Kloss et al., 1999; Verdier and Penke, 2004; Wallquist et al., 2004). Using integrin antagonists as therapeutic agents in a variety of neurological diseases is of great interest at present (Blackmore and Letourneau, 2006; Mattern et al., 2005; Polman et al., 2006; Wang et al., 2006). In this chapter, we describe methods used in our laboratory to characterize neuronal responses to extracellular matrix proteins, and procedures for assessing integrin roles in neuronal cell attachment and differentiation.

Introduction

In this section, we provide a brief summary of the current state of our knowledge on the roles of integrin receptors in the nervous system, beginning with their roles in neural development. Since the 1980s, the many roles for this fascinating family of adhesion molecules have been documented, which has increased our understanding of neural development, synaptic function, and several neurological diseases.

Much of the initial interest in integrin roles in the nervous system was focused on their roles in the neural crest, a migratory population of cells derived from the dorsal neural tube that populate the sensory, autonomic, and enteric nervous systems as well as contributing to formation of many specialized sense organs, glands, the heart, cranial mesenchyme and bone, and supporting cells in peripheral nerves, including Schwann cells and endoneural fibroblasts (Le Douarin and Dupin, 2003). Neural crest cells express many integrins and migrate through an extracellular matrix (ECM)‐rich environment (Bronner‐Fraser 1994, Kil 1998). Acute inhibition experiments in avian embryos have documented important roles for integrins in migration of the neural crest (Tucker, 2004). In mice, genetic ablation of β1 integrins results in severe perturbations of the peripheral nervous system, including failure of normal nerve arborization, delay in Schwann cell migration, and defective neuromuscular junction differentiation (Pietri et al., 2004). In addition to direct effects on migration, it has been shown that absence of specific integrin heterodimers compromises Schwann cell precursor survival, proliferation, and differentiation (Feltri 2002, Haack 2001). Many of these observations are likely to reflect the roles of integrin receptors in regulating activation of MAP kinase, Rac, and other signaling pathways (Campos et al., 2004).

In part because they were characterized long before the identification of the major families of axon guidance molecules, such as the netrins, semaphorins, and ephrins, early studies on axon outgrowth and guidance focused on integrins and the cadherin and immunoglobulin families of cell adhesion molecules. In these studies, it was demonstrated that virtually all process extension on ECM substrates by neurons requires integrin function and that neuronal growth cones could distinguish between different ECM proteins and respond to orientation or gradients of ECM proteins by directed growth (Dubey 1999, McKenna 1988). In addition, integrins have been shown to interact with several of the axon guidance systems. For example, semaphoring 7A‐dependent promotion of axon growth requires integrin activity, while semaphoring‐mediated activation of plexin signaling reduces integrin‐based adhesion (Barberis 2004, Pasterkamp 2003). The A and B ephrins also control integrin activation (Davy 2000, Nakada 2005). Integrins interact genetically with the Slit‐Robo pathway in Drosophila (Stevens and Jacobs, 2002). Evidence also suggests that two integrins may serve as receptors for netrins in epithelia (Yebra et al., 2003). Despite these intriguing observations, ablation of either β1 or αV integrins appear to have only minor effects on axon guidance in the brain although perturbations are most significant in the peripheral nervous system (Blaess 2004, Graus‐Porta 2001, McCarty 2005, Proctor 2005). This is likely the result of neurons interacting with many different types of substrates, many of which do not require integrin function.

Despite the absence of major effects on axon guidance, integrin deletion affects many aspects of forebrain and cerebellar development. First, loss of β1 integrins results in disruptions of the basal lamina that separates the brain from the overlying mesenchyme (Graus‐Porta et al., 2001). As a result, the migration of neurons is perturbed, resulting in abnormal lamination of the cortex and cerebellum. Similar phenotypes are observed in mice lacking another ECM receptor dystroglycan as well as in humans and mice with mutations in basal lamina–encoding genes, such as the laminin α5 subunit (Gleeson and Walsh, 2000). Although some evidence indicates that integrins modulate neuronal interactions with radial glia—which provide the substrate for the tangential migrations that establish the cortical lamination pattern (Sanada 2004, Schmid 2005)—the major phenotype observed in these mutants appears to stem from disruption of signaling pathways controlling neuronal migration that require integrity of the basal lamina (Beggs et al., 2003).

Integrins have a number of additional actions that modulate brain development. Of particular interest, they have been shown to control survival and proliferation of some populations of neural stem cells (Leone et al., 2005). Within the external granule cell layer of the cerebellum, integrin binding to laminin enhances the proliferative responsiveness of granule cells to sonic hedgehog (SHH), probably because association of SHH with laminin facilitates SHH activation of its receptor Smoothened (Blaess et al., 2004).

Integrins have a number of potent, but poorly understood, effects on synaptic function and plasticity. In cell culture, interactions of astroglia with neurons mediated by the glial integrin αVβ3 results in PKC activation in individual neurons that facilitates excitatory synaptogenesis (Hama et al., 2004). The receptor on neurons mediating PKC activation is not known, but neurons express many proteins, including the ADAMs, L1, and amyloid precursor protein, which are known to interact with integrins and are therefore candidates to mediate this signaling pathway (Mechtersheimer 2001, Wright 2006, Yang 2006). In Drosophila, integrins control localization of postsynaptic proteins at the neuromuscular junction through a CamKII‐mediated signaling cascade (Burgess et al., 2002). At the vertebrate neuromuscular junction, expression of integrins in muscle, but not nerve, is required for synapse formation (Schwander et al., 2004), possibly through interactions with agrin or promotion of basal lamina assembly/organization (Burgess 2002, Burkin 2000). Integrins have also been localized to the synaptic active zones of motor neuron axon terminals and mediate the enhancement of transmitter release caused by mechanical stretching of muscle fibers (Kashani et al., 2001).

Although localization studies indicate that integrins are present at many synapses in the brain, genetic and pharmacological studies indicate that integrins are not required for synapse formation, but are required for normal synaptic plasticity. In a particularly elegant series of studies, the presence of integrins in the mushroom body of the Drosophila brain was shown to be required for short‐term memory (Grotewiel et al., 1998). Conditional expression of an integrin subunit in the adult mushroom body rescued the memory deficits, providing definitive evidence that this was an effect on function, not early development. Studies in the murine hippocampus have demonstrated that β1 integrins are required for normal LTP (Chan 2006, Huang 2006). Studies of mice with reduced expression of individual β1 integrin heterodimers have suggested that specific integrins have different functions at the synapse (Chan et al., 2003). Acute pharmacological perturbations using inhibitory integrin reagents indicate that integrins are involved in regulation of both NMDA and AMPA receptor function and act through regulation of protein kinases and the actin cytoskeleton (Kramar 2003, Kramar 2006, Lin 2003). Clearly, much remains to be understood about interactions between integrins and the signaling pathways known to be fundamental in initiation and maintenance of LTP.

Integrins are also necessary for normal development of non‐neuronal cells in the nervous system, including astroglia, oligodendrocytes, and Schwann cells. For example, the presence of β1 integrins is required for normal morphological development of radial glia, and abnormalities in the morphology of these glia may underlie the neuronal lamination deficits observed in mice with mutants in genes encoding basal lamina constituents and their receptors, including both integrins and dystroglycan (Forster 2002, Gleeson 2000).

An abundance of studies in culture indicate that integrins have profound effects on the differentiation of the precursors to oligodendrocytes (Colognato 2002, Colognato 2004). Despite these effects of integrin absence on oligodendrocyte development and myelination in the brain and spinal cord appear to be modest. In mice lacking β1 integrins myelination appears to be completely normal during development and, following injury, in the adult (Benninger et al., 2006). During development there is elevated apoptosis of premyelinating oligodendrocytes, but this does not prevent successful myelination. No obvious reduction in myelination is obvious in mice lacking either the αV or β8 integrins, although this has been examined in the same detail as the studies performed using mice lacking the β1 integrin subunit (McCarty 2005, Proctor 2005).

β1 integrins are clearly essential for many steps in peripheral nerve development, including several aspects of Schwann cell differentiation. β1 gene ablation in the neural crest cell lineage delayed the migration of Schwann cell precursors along embryonic peripheral nerves without detectable effects on proliferation or apoptosis (Pietri et al., 2004). At later times, the absence of these integrins interfered with normal sorting of sensory axons with Schwann cells and reduced their myelination (Feltri 2002, Pietri 2004). Integrin deficiency in the neural crest cell lineage also interfered with basal lamina assembly in peripheral nerves and prevented normal differentiation of the neuromuscular junction. No obvious phenotypes in the peripheral nervous system have been reported in mice lacking the αV or β8 integrins.

Absence of either αV or β8 integrins does, however, have a profound influence on brain development. Expression of αVβ8 is required for normal vascular development in the brain. In its absence, vascular development is severely perturbed, resulting in massive embryonic hemorrhage. Cell‐specific targeting has shown that this integrin must be expressed in the neuroepithelial, not the endothelial lineage (McCarty 2005, Proctor 2005). Intriguingly, αVβ8 has been shown to promote the activation of TGFβ through binding to an RGD sequence in the TGFβ latency‐associated peptide (Mu et al., 2002). Similarities between the phenotypes of the αV and β8 and mutations in the TGFβ signaling pathway suggest that the vascular abnormalities may reflect, at least in part, a reduction in TGFβ activation. Surprisingly, in animals that survive hemorrhage, the vasculature recovers so that hemorrhage is no longer visible in young adults. These animals, however, do develop motor deficits and die prematurely, most likely as a result of neurodegeneration in the central nervous system (CNS).

Integrins are also of interest to neuroscientists because they have been implicated in several neurodegenerative disorders. Of particular importance, because integrins regulate the transit of lymphocytes, macrophage, and other cells across the blood–brain barrier in response to inflammatory stimuli, anti‐integrin reagents are of great interest as therapeutic agents to control demyelinating diseases, such as multiple sclerosis that involve the immune system and inflammatory responses (Bartt 2006, Kanwar 2000). The involvement of integrins in inflammatory responses has created interest in the possibility of using anti‐integrin reagents to alleviate several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases (Austin et al., 2006). Of special interest, several integrins appear to interact with amyloid precursor protein and these are postulated to mediate deposition or toxic actions of Aβ and amyloid formation (Bozzo 2004, Koenigsknecht 2004, Sondag 2006). Thus future studies on integrin functions in the normal and diseased brain are likely to provide interesting insights, some of which may have practical applications.

Section snippets

Standard substrate preparation

When substrates such as laminin, fibronectin and the collagens, are available in abundant quantities, substrates are typically prepared in the same way as for other cell types by incubation of substrata with ECM proteins in solution, washing, and blocking, and then neuronal adhesion or neurite outgrowth assays. For example, the laboratory has frequently used the following protocol (Hall et al., 1987) in which sterile Linbro 96‐well, flat‐bottom tissue culture plates are coated with 100 μl per

Neuronal Culture Procedures

Procedures used by our laboratory for isolation and culture of several different types of neurons are described below. These assays typically give enriched, not absolutely pure populations of neurons. For studies where highly purified populations of neurons are required, antibodies specific for different cell types have been used in sequence to deplete cell mixtures of unwanted cell types and to select for specific subpopulations of neurons, such as retinal ganglion cells (Barres 1999, Goldberg

Biochemical Studies Using Cultured Neurons

Purified neuronal populations are typically present in limited quantities, so sensitive assays are required to detect integrins and other proteins. It is absolutely essential to assess the purity of a culture before making conclusions about cell type–specific expression of integrins or other proteins because non‐neuronal cells (both astroglia and endothelial cells that contaminate CNS cultures and Schwann cells and fibroblasts, contaminants of peripheral neuronal cultures) express many

References (78)

  • H. Haack et al.

    Integrin receptors are required for cell survival and proliferation during development of the peripheral glial lineage

    Dev. Biol.

    (2001)
  • H. Hama et al.

    PKC signaling mediates global enhancement of excitatory synaptogenesis in neurons triggered by local contact with astrocytes

    Neuron

    (2004)
  • E. Hawrot et al.

    Long‐term culture of dissociated sympathetic neurons

    Methods Enzymol.

    (1979)
  • J.R. Kanwar et al.

    Beta7 integrins contribute to demyelinating disease of the central nervous system

    J. Neuroimmunol.

    (2000)
  • S.H. Kil et al.

    The alpha4 subunit of integrin is important for neural crest cell migration

    Dev. Biol.

    (1998)
  • E.A. Kramar et al.

    Integrins modulate fast excitatory transmission at hippocampal synapses

    J. Biol. Chem.

    (2003)
  • N.M. Le Douarin et al.

    Multipotentiality of the neural crest

    Curr. Opin. Genet. Dev.

    (2003)
  • M.P. McKenna et al.

    Growth cone behavior on gradients of substratum bound laminin

    Dev. Biol.

    (1988)
  • A. Meyer‐Franke et al.

    Depolarization and cAMP elevation rapidly recruit TrkB to the plasma membrane of CNS neurons

    Neuron

    (1998)
  • M. Nakada et al.

    EphB2/R‐Ras signaling regulates glioma cell adhesion, growth, and invasion

    Am. J. Pathol.

    (2005)
  • K.M. Neugebauer et al.

    Vitronectin and thrombospondin promote retinal neurite outgrowth: Developmental regulation and role of integrins

    Neuron

    (1991)
  • K. Sanada et al.

    Disabled‐1–regulated adhesion of migrating neurons to radial glial fiber contributes to neuronal positioning during early corticogenesis

    Neuron

    (2004)
  • K.J. Tomaselli et al.

    A neuronal cell line (PC12) expresses two beta 1‐class integrins—alpha 1 beta 1 and alpha 3 beta 1—that recognize different neurite outgrowth‐promoting domains in laminin

    Neuron

    (1990)
  • R.P. Tucker

    Antisense knockdown of the beta1 integrin subunit in the chicken embryo results in abnormal neural crest cell development

    Int. J. Biochem. Cell Biol.

    (2004)
  • B. Varnum‐Finney et al.

    The integrin receptor alpha 8 beta 1 mediates interactions of embryonic chick motor and sensory neurons with tenascin‐C

    Neuron

    (1995)
  • C. Xie et al.

    Survival of hippocampal and cortical neurons in a mixture of MEM+ and B27‐supplemented neurobasal medium

    Free Radic. Biol. Med.

    (2000)
  • P. Yang et al.

    The ADAMs family: Coordinators of nervous system development, plasticity and repair

    Prog. Neurobiol.

    (2006)
  • M. Yebra et al.

    Recognition of the neural chemoattractant netrin‐1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration

    Dev. Cell

    (2003)
  • S.A. Austin et al.

    Alpha‐synuclein expression modulates microglial activation phenotype

    J. Neurosci.

    (2006)
  • D. Barberis et al.

    Plexin signaling hampers integrin‐based adhesion, leading to Rho‐kinase independent cell rounding, and inhibiting lamellipodia extension and cell motility

    FASEB J.

    (2004)
  • B.A. Barres et al.

    Axonal control of oligodendrocyte development

    J. Cell Biol.

    (1999)
  • R.E. Bartt

    Multiple sclerosis, natalizumab therapy, and progressive multifocal leukoencephalopathy

    Curr. Opin. Neurol.

    (2006)
  • Y. Benninger et al.

    Beta1‐integrin signaling mediates premyelinating oligodendrocyte survival but is not required for CNS myelination and remyelination

    J. Neurosci.

    (2006)
  • S. Blaess et al.

    Beta1‐integrins are critical for cerebellar granule cell precursor proliferation

    J. Neurosci.

    (2004)
  • M. Bronner‐Fraser

    Neural crest cell formation and migration in the developing embryo

    FASEB J.

    (1994)
  • V.L. Buchman et al.

    Different neurotrophins are expressed and act in a developmental sequence to promote the survival of embryonic sensory neurons

    Development

    (1993)
  • R.W. Burgess et al.

    Mapping sites responsible for interactions of agrin with neurons

    J. Neurochem.

    (2002)
  • D.J. Burkin et al.

    Laminin and alpha7beta1 integrin regulate agrin‐induced clustering of acetylcholine receptors

    J. Cell Sci.

    (2000)
  • L.S. Campos et al.

    Beta1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance

    Development

    (2004)
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