Laminin and the mechanism of neuronal outgrowth

Brain Research Reviews 23 Ž1997. 1–27

Full-length review

Laminin and the mechanism of neuronal outgrowth )

Louise Luckenbill-Edds

Department of Biological Sciences and College of Osteopathic Medicine, Ohio UniÕersity, Athens, OH 45701, USA Accepted 30 July 1996

Abstract This review summarizes the structure of the laminin molecule and the role it plays in development, pathfinding and regeneration in the vertebrate nervous system. Laminin has proven to be an influential glycoprotein of the extracellular matrix which guides and promotes the differentiation and growth of neurons. Its numerous domains, its association with carbohydrate moieties, and its many isoforms associated with specific sites and stages will be important in elucidating its function. How laminin’s signals become translated into changes in the behavior of cells remains one of the thorniest issues facing scientists working at the interface between neuronal growth cone and extracellular matrix. New approaches using molecular biological tools and immunological tools for dissecting the laminin molecule have provided hints of intramolecular shifts in laminin’s properties which influence cell behavior. These shifts occur in response to other molecules in the extracellular matrix such as carbohydrates, or in response to moieties on the cell surface itself. Thus, reduction of laminin’s structure to fragments and ultimately polypeptide sequences is leading to renewed significance of laminin’s tertiary and quaternary structure with respect to laminin’s biological interactions. Such insights about laminin’s structure are providing new tools for probing growth cone behavior, tools that need to be coupled with equally sophisticated analyses of growth cone behavior using biophysical and biochemical measures at a biological level suitable for analyzing responses induced by the probes. Keywords: Laminin; Laminin isoform; Integrin receptor; Growth cone; Neuronal pathfinding; Synapse formation; Alzheimer’s disease; Nerve regeneration

Contents 1. Introduction

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2. Structure and isoforms .

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3. Assembly and polymerization in basement membrane 4. Binding and signaling . . . 4.1. Signaling mechanisms 4.2. Binding to receptors .

5. Mechanism of promotion of neurite outgrowth .

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7. Laminin in developing brain

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8. Laminin and Alzheimer’s disease .

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6. Laminin in growth cone guidance, pathfinding, and synapse formation 6.1. Guidepost model . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Patterned substrate model . . . . . . . . . . . . . . . . . . . . . 6.3. Laminin-3 in synapses . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author. Fax: q1 Ž614. 593-0300; E-mail: [email protected]

0165-0173r97r$32.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 1 7 3 Ž 9 6 . 0 0 0 1 3 - 6

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L. Luckenbill-Eddsr Brain Research ReÕiews 23 (1997) 1–27

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9. Laminin in peripheral regeneration and central nervous system 9.1. Peripheral nerve regeneration . . . . . . . . . . . . . . . 9.2. Central nervous system . . . . . . . . . . . . . . . . . . 10. Summary .

Acknowledgements . References

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1. Introduction Laminin is a large Ž Mr s 850,000., multidomained, cross-shaped glycoprotein that is organized in the meshwork of basement membranes, such as those lining epithelia, surrounding blood vessels and nerves, and underlying pial sheaths of the brain w83x. It also occurs in the extracellular matrix ŽECM. in sites other than basement membranes at early stages of development, and is localized to specific types of neurons in the central nervous system ŽCNS. during both embryonic and adult stages. Synthesized and secreted by cells into their extracellular environment, laminin in turn interacts with receptors at cell surfaces, an interaction that results in changes in the behavior of cells such as attachment to a substrate, migration, and neurite outgrowth during embryonic development and regeneration. Information about the laminin molecule is based on a number of techniques, including controlled proteolysis followed by electron microscopy of rotary shadowed molecules, sequencing of the polypeptide chains and cloning recombinant fragments, immunocytochemistry for localizing it in tissues, and cultures of cells and tissues for studying its interaction with cells. Laminin has been investigated in the basement membrane of a mouse embryonal carcinoma-derived cell line by Chung et al. w15x, and also in the murine Engelbreth–Holm–Swarm ŽEHS. tumor which synthesizes and secretes large amounts of basement membrane components, 50% of which by weight is laminin w35,84x. Although the large harvests of basement membrane from the EHS tumor have resulted in much of our classic information about laminin, recent results from cDNA sequencing hold promise for the emergence of new, detailed information on the structure of laminin. For example, several variants of laminin have been discovered in different species and tissues, leading to the concept of the heteromeric assembly of structurally different polypeptide chains into laminin isoforms. Such isoforms reflect not only adaptation to different functions, but also a complex phylogeny of the molecule.

2. Structure and isoforms Laminin is composed of three polypeptide chains: an a chain Žformerly A, 400 kDa., a b chain Žformerly B1, 210

kDa., and a g chain Žformerly B2, 200 kDa. ŽFig. 1. w13,82x. The three chains are arranged in a cross whose two short arms consist of either b or g chains and whose long arm consists of the a chain plus parts of each b and g chain ŽFig. 1.. The polypeptide chains of laminin are

Fig. 1. Structural model of laminin. Chains designated by Greek letters, domains by Roman numerals. cys-rich rod domains in the short arms are designated by symbols S, the triple coiled-coil region Ždomain I–II. of the long arm by parallel straight lines. In the b1 chain, the a-helical coiled-coil domains are interrupted by a small cys-rich domain a. Interchain disulfide bridges are indicated by thick bars. Regions of the molecule corresponding to fragments E1X, 4, E8, and 3 are indicated. G1–G5 are domains within the terminal globule of the a1 chain. Modified from Beck et al. w5x, with permission from the Federation of American Societies of Experimental Biology.

L. Luckenbill-Eddsr Brain Research ReÕiews 23 (1997) 1–27

composed of domains with characteristic sequences and tertiary structure, for example, globular domains at the carboxy terminal of the a chain and within the short arms; a-helical domains at the amino termini of the b and g chains; rod-like domains with b-pleated structure, and epidermal growth factor-like ŽEGF. repeats with cystein motifs in all three chains ŽFig. 1.. The multi-domain structure of laminin means that specific domains are associated with distinct functions such as cell attachment, promotion of neurite outgrowth, and binding to other glycoproteins or proteoglycans. Within these domains specific fragments of polypeptide sequences, as few as several amino acids, have been identified with specific biological activity. For example, two different peptide sequences, IKVAV and LQVQLSIR within the E8 domain on the long arm function in cell attachment and promote neurite outgrowth ŽFig. 1. w81,66x. About 25–26% Žweightrweight. of laminin consists of carbohydrate moieties that are unusual in their variety and particularly rich in galactosyl residues in the N-oligosaccharides w19x. These moieties, found at the surface of the coiled coil of the laminin molecule, are important in biological function of laminin, including neurite outgrowth. When PC-12 cells are plated on laminin substrates whose glycosyl residues are either blocked with the lectin conconavalin A or are absent Žnon-glycosylated laminin., then promotion of neurite growth is impaired, although cell attachment appears not to be affected w19x. The glycosyl moieties per se seem to be involved here, since circular dichroism studies of glycosylated and non-glycosylated indicate no differences in secondary structure, the eliminating the possibility that the carbohydrate acts by influencing the secondary structure of laminin w19x. Laminin also binds purified heparan sulfate proteoglycan, in an interaction between heparan sulfate side chains and the two most terminal globular G domains of fragment E3 in the a1 chain w4,96x. Additional studies have characterized the heparan sulfate proteoglycan as perlecan w18x. Perlecan, representing approximately ten percent of the

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EHS basement membrane, is a proteoglycan with two major forms, one with only heparan sulfate chains, the other a hybrid molecule with dermatan sulfate chains as well, in addition to small amounts of forms with other glycosaminoglycans or none at all w18x. A second proteoglycan in EHS tumor matrix is basement membranechondroitin sulfate proteoglycan Žbamacan. containing chondroitin sulfate. Each proteoglycan has distinctive core protein chains and can bear galactosaminoglycan residues w18x. Variants of EHS laminin include laminins with different polypeptide chain composition Ž a1, a 2, a 3; b1, b2, b3; g1, g2. assembled into heterotrimeric isoforms w82x. All forms discovered up to now are trimers that include one of each of the three groups of polypeptide chains ŽTable 1., although heterodimers of the b and g chains have been observed in vitro. The EGF-like repeats in the short arms of all three polypeptide chains are consistently found in 60% of the laminin isoforms examined, whereas the rodlike region of the long arm that contains all three chains is the most variable region in different isoforms. The relatively large number of variant polypeptide sequences and trimer composition Žno doubt their number will increase. means that the domains of laminin as well as its three-dimensional structure have been modified for specific functions and locations within tissues among different species. Several studies localizing laminin isoforms or their mRNAs produced within peripheral and central nervous system tissues have been carried out. For example, human fetal tissues Ž18–19 weeks. have been subjected to in situ hybridization techniques to detect the expression of mRNA for laminin chains with the following results: mRNA for the a1 chain is expressed in brain, especially in neuroretina, olfactory bulb, and cerebellum, meninges, as well as in kidney and testis, whereas mRNA for the a 2 chain is expressed strongest in kidney, heart, skin and lungs, i.e., in mesenchyme-derived cells w89x. In situ hybridization techniques with adult rat dorsal root ganglia using probes from nonhomologous regions of each laminin chain localized g1

Table 1 Laminin isoforms Isoform

Location

Chains

Other

Laminin-1 Laminin-2 Laminin-3 Laminin-4 Laminin-5 Laminin-6 Laminin-7 Laminin-8 d Laminin-9 d Laminin-10 d

EHS tumor cardiac, skeletal muscle; nerve neuromusc. junction; renal glomeruli Schwann cells; skeletal muscle dermo-epidermal junction dermo-epidermal junction human amnion human chorion human chorion human amnion, keratinocyte cultures

a1,b1,g1 a 2,b1,g1 a1,b2,g1 a 2,b2,g1 a 3,b3,g2 a 3,b1,g1 a 3,b2,g1 a4,b1,g1 a4,b2,g1 a 5,b1,g1

prototype, embryonic type, mammal, fruitfly merosin a s-laminin a s-merosin a kalinin or nicein, short arm deletions b k-laminin c k-laminin c y y y

a b c d

Cross-shaped molecule. Short a 3 chain results in Y-shaped molecule. Proteolytic processing of all chains. Champliaud, M.-F. and Burgeson, R.E., personal communication.

a

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L. Luckenbill-Eddsr Brain Research ReÕiews 23 (1997) 1–27

mRNA in neurons, satellite cells, and Schwann cells, b1 mRNA in satellite and Schwann cells, but no a1 chain mRNA in any of the cell types. Satellite cells and Schwann cells also exhibited mRNA for a 2 Žmerosin. mRNA w40x. Immunofluorescence staining of the connective tissue of human peripheral nerve revealed that a1, b2, and g1 chains are present in perineurium, whereas a 2, b1, b2, and g1 chains are present in endoneurium. If cells from these tissues are cultured, however, they produce all five chains, indicating a ‘plasticity’ of expression that may depend on interactions in organized tissue w30x. Laminin-3 Ž a1b2 g1., also localizes at neuromuscular synapses, whereas laminin-1 Ž a1b1g1. and laminin-2 Ž a 2b1g1. isomers occur in extrajunctional basement membrane of skeletal muscle cells w71x. Besides varying in expression of laminin isoforms, neurons also vary in their responses to various isoforms. Five neuronal cell lines differentially respond to different laminin isoforms with respect to adhesion and neurite outgrowth on a number of synthetic peptides prepared from a1 and a 2 chains of laminin w66x.

3. Assembly and polymerization in basement membrane Since several isoforms can assemble from heterotrimers with variant polypeptide chains, this means that the association of the three chains depends on a selective mechanism that is independent of the specific variant polypeptide sequences. The long arm of laminin is important, since unfolding this region by treating with urea leads to reversible dissociation of the triple-stranded coiled-coil structure w5x. Evidence from experiments on heterotrimer formation using short recombinant polypeptide fragments of a 2, b1, and g1 chains points to a critical role for the C-terminal region of the long arm in chain selection during assembly. Specifically, the isoleucine residues of the a 2 and g1 chains are essential for stabilizing the native-like, triple-stranded structure of laminin w62x. A two-step sequence of assembly is proposed, with b and g chains forming heterodimers first, followed by association with the a chain w86x. Thus, the N-terminal domains of all three chains would be involved in polymerization. Support for this is based on experiments with a recombinant a1 Nterminus chain Žra 1ŽVI–IVb.. which inhibits polymerization in a concentration-dependent manner, a result interpreted as depending on ‘‘parallel, separate contributions from each individual short arm’’ w17x. Polymerization, the higher order assembly of laminin into the basement membrane, depends on time, temperature, and concentration w95x. Initial oligomer formation requires Ca2q to induce a required conformational change and polymerization proceeds by non-covalent interaction of the outer globules of the short arms ŽFig. 2.. About 80% of laminin in the basement membrane exists in a self-assembled network, whereas the other 20% is anchored to a

collagen ŽIV. network either directly or through interactions with entactinrnidogen, a minor glycoprotein in the basement membrane ŽFig. 2. w82x. Varying ratios of laminin and collagen ŽIV. can confer different properties to meshworks of basement membranes, since laminin networks are lattice-like and reversible, whereas collagen ŽIV. networks are polygonal and more stable ŽFig. 2. w95x. Additional variations within the meshwork of the basement membrane are possible when one includes isoforms of the polypeptide chains Žsee above., post-translational modification of oligosaccharides associated with laminin or collagen ŽIV., and the synthesis of site-specific components in different tissues and species. The production of recombinant chains has already provided new insights about the importance of the quaternary structure of laminin for binding to associated molecules like heparan sulfate proteoglycan and for interacting with cell surfaces w80x. In this study, a hybrid E8 fragment was assembled by intercalating recombinant a1 chains with isolated b1 and b2 chains that had been purified from authentic ŽEHS tumor. fragment E8 Žsee Fig. 1.. Thus, the hybrid glycoprotein ŽB-rAiG. consisted of the long arm of laminin with rod and globule N-terminal ŽFig. 1. whose a1 chain had been secreted by baculovirus and whose b1 and g1 chains had been purified using chaotropic agents like 8M urea. The properties of this hybrid E8 fragment were compared with the properties of the recombinant a1 chains alone, or with a1 or b1 g1 chains isolated from fragment E8 w80x. The hybrid glycoprotein did not bind as strongly to heparan sulfate proteoglycan as did isolated G domains or the recombinant a1 chains, indicating that b1 and g1 chains may suppress binding of the G domain to heparan w80x. When adhesion of HT1080 cells was examined, cells did not adhere to a1 chains or b1 and g1 chains isolated with urea from E8, but did adhere to rAiG and to B-rAiG as long as the recombinant chains were not urea-treated. Adhesion of cells to B-rAiG, however, could be blocked by monoclonal antibodies to a6 and b1 integrins, whereas adhesion to rAiG was not blocked by these antibodies. This indicates that adhesion to rAiG is mediated by another a receptor besides the a6 and b1 integrins and that the ligand in authentic laminin a1 chain is destroyed by urea treatment. Thus new functions of the laminin a1 chain that are dependent on the tertiary or quaternary structure have been revealed in these investigations w80x, supporting similar conclusions drawn from previous investigations of the E8 fragment using proteolysis and denaturation to produce fragments w20x.

4. Binding and signaling A number of receptors and binding proteins regulate the interaction between laminin and the cell surface w56x. These receptors and binding proteins exhibit varying degrees of binding affinity, indicating that several types of

L. Luckenbill-Eddsr Brain Research ReÕiews 23 (1997) 1–27

interactions with laminin may occur simultaneously on the cell surface. Not only does one ligand such as laminin interact with several receptors, but also some receptors are ‘promiscuous’ and interact with more than one ligand in the ECM. In addition to the spatial organization of receptors for laminin on cell surfaces, there may also be temporal organization so that one or another receptor plays a role at different times during neurite outgrowth. In summarizing this complex situation regarding cell surface receptors for laminin, Begovac et al. w6x suggest that no single receptor is solely responsible for mediating laminin’s promotion of neurite outgrowth, rather there may be hierar-

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chies of importance as well as cooperativity among mechanisms. 4.1. Signaling mechanisms The transmembrane location of many receptors means that they communicate information from ECM to cytoskeletal machinery involved in modulating cell behavior such as spreading, adhesion, and neurite outgrowth. Circumstantial evidence for signaling pathways is based on preparations of growth cone particles from embryonic brain which contain high levels of the intracellular tyrosine

Fig. 2. A working hypothesis of basement membrane assembly and structure. Panel A: Laminin Ž1. forms terminal-domain-associated oligomers and then Ž2. forms polymers in the presence of calcium. The low-density Žhigh molecular weight. heparan sulfate proteoglycan ŽHSPG. binds to itself at a core pole opposite the origin of the HS chains, and the HS chains bind to the long-arm globules of laminin. Laminin also polymerizes with type IV collagen Žpanel B., binding to collagen at a site near the C-terminus, becoming entrapped in the collagen network. Entactin Žnidogen. and heparan sulfate may serve as a bridge between laminin to collagen Žnot shown in model.. Panel C: Laminin in tethered complexes with HSPG binds to collagen ŽIV., or forms an independent network within the basement membrane. Modified from Yurchenco w95x, with permission of the New York Academy of Science.

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L. Luckenbill-Eddsr Brain Research ReÕiews 23 (1997) 1–27

kinase pp60c-arc and the growth-associated protein GAP43, both implicated in signaling w10x. However, to show which signaling mechanisms are responsible for growth cone behavior during neurite extension on laminin, investigators have turned to primary cultures of neurons or neuronal cell lines and have used three basic approaches for understanding the mechanisms involved in response to ECM w10x: Ž1. neurons are grown on a purified substrate of the molecule under study and probed with pharmacological agents to either inhibit or enhance the signaling system involved in neurite outgrowth; Ž2. neurons are grown on substrates of the molecule under investigation and specific antibodies are added to block to function of the molecule; and Ž3. neurons are transfected with genes for the growthpromoting molecule, or alternatively are treated with antisense mRNA to deplete the intracellular stores of the molecule, and cell behavior is assessed. Despite many of these types of studies, the signaling system downstream following the interaction of laminin with receptor or binding protein remains unclear. On the one hand, evidence based on neurons cultured for 16–20 h shows that laminin-mediated neurite outgrowth may involve a protein kinase C system ŽPKC. which phosphorylates signaling molecules. In these experiments phorbol ester 12-O-tetradecanoylphorbol-13-acetate ŽTPA., an activator of this kinase, promotes neurite outgrowth when chick ciliary ganglion neurons are cultured on suboptimal concentrations Žapprox. 1 mgrml. of laminin substrate for 16–20 h. Inhibitors of this kinase, H7 Žan isoquinoline sulfonamide. and sphingosine, inhibit outgrowth on optimum concentrations Žgreater than 2 mgrml. of laminin w9x. Thus, laminin-stimulated process outgrowth in relatively longterm cultures of neurons appears to involve protein phosphorylation. Additional evidence for protein kinase C involvement in neurite outgrowth and growth cone adhesion is based on experiments with the growth-associated protein GAP-43 w1x. GAP-43 is a major protein kinase C substrate that is acylated reversibly and is localized near the plasma membrane of growth cones and associated with the cortical cytoskeleton. Experiments on the role of GAP-43 in growth cone extension will be reviewed at the end of Section Section 5. On the other hand, evidence from NG108-15 Žan astroglial-neuronal hybrid cell line. and PC12 Ža pheochromocytoma cell line. cell lines cultured on higher concentrations Ž10 mgrml. of laminin substrate for 3 h shows that dephosphorylation is increased, whereas phosphorylation is reduced and TPA inhibits laminin-mediated process outgrowth w92x. Also, since laminin stimulates dephosphoryation of several laminin-binding protein Ž110 kDa, 67 kDa. and enhancers of phosphorylation by kinases inhibit process outgrowth, it appears that early events in signal transduction in these cell lines involve dephosphorylation w92x. Many more experiments need to be carried out using different neuronal cell lines and different phases of process outgrowth to clarify the role of laminin in signaling by

means of either phosphorylation or dephosphorylation, or both. It could turn out that different signaling pathways are used to mediate different, as yet unknown behaviors, during process outgrowth. 4.2. Binding to receptors Several techniques have been used to investigate interactions between cell surface receptors and laminin: affinity chromatography of extracts of cell membranes with domains or peptide sequences of laminin, blockage of receptor-mediated cell behavior with antibodies to receptors or binding proteins, and transfer of genes for receptor subunits by transfecting cells with polypeptide subunits of various integrin receptors. The types of receptors that bind laminin to cell surfaces and mediate adhesion and neurite outgrowth include: Ž1. Integrins of the b family; Ž2. Non-integrin binding proteins that bind specific sequences in one of the polypeptide chains; and Ž3. Carbohydrate-binding moieties such as lectins that bind galactose and poly-N-acetyllactosamine groups in oligosaccharides, and galactosyltransferase, an enzyme that binds to laminin Žfor a review of ECM receptors and neurons, see w67x.. 4.2.1. Integrin receptors Integrins are heterodimeric Ž ab ., divalent cation-dependent receptors with affinity for a number of extracellular matrix ŽECM. components. The current total of 15a and 8b subunits creates a family of at least 20 different heterodimers. The best characterized members of this family which mediate the binding of neurons and neuronal cell lines to laminin include a 3b1 and a6b1 which bind to the E8 fragment in the laminin a1 chain of the long arm, and a1b1 which binds to the E1 fragment of the short arm and to recombinant mouse laminin a1 chain that contains domains VI–IVb Žra1ŽVI–IVb.X . w17x. Integrin b8 has also been demonstrated to be important for adhesion and neurite outgrowth of embryonic chick sensory neurons on laminin-1, collagen IV, and fibronectin substrates w87x. Some integrin receptors recognize the same peptide sequence in different ECM molecules, hence a given integrin may bind to laminin, collagen, and fibronectin w85x. Also, a given neuronal cell type may use more than one integrin receptor to interact with an ECM substrate in neurite outgrowth. For example, neurite outgrowth could be only partially inhibited with antibodies to either b1 or b8 alone when embryonic chick sensory neurons were cultured on a fibronectin substrate, whereas combined antibodies to these integrin subunits completely blocked outgrowth w87x. In the authors’ view this means that more than one integrin b subunit, in addition to one or more a subunits are involved in laminin-mediated neurite outgrowth from these neurons w87x. Integrin receptors are important for cell adhesion as well as neurite outgrowth, as shown for the dominant integrin receptor of PC12 cells for laminin, a1b1, which

L. Luckenbill-Eddsr Brain Research ReÕiews 23 (1997) 1–27

binds domain VI from the short arm of laminin-1 w17x. This was demonstrated by using a polyclonal antibody to whole laminin to block neurite outgrowth on a laminin substrate, and additionally showing that absorption of the antibody with domain VI restores adhesion Žand neurite outgrowth. of PC12 cells to the laminin substrate. The interpretation is that domain VI competitively binds to the site in the antibody needed to block integrin-mediated adhesion of PC12 cells on laminin w17x. The integrin receptors are ‘integral’ components of the plasma membrane with a carboxy terminal that interacts with cytoskeletal elements in the cytoplasm and an amino terminal which interacts with ECM components. If PC-12 cells are grown on a laminin substrate, prepared as cytoskeletal ghosts, then immunocytochemically labeled for integrin subunits, the ghosts display integrins a1b1 in a punctate pattern in focal contacts on the surfaces exposed to the laminin substrate ŽFig. 3. w2x. The a 3 subunit is not associated with the cytoskeleton and its immunoreactivity is lost following immunocytochemical processing. When extracted with detergent, half of these cells’ integrin subunits of the a1 and b1 types are retained with the cyto-

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skeletal components actin and tubulin. On the other hand, when grown in suspension, the a1 and b1 integrin subunits are solubilized by detergent and are not retained with the cytoskeleton. Integrin a 3 is always solubilized. These results suggest that cell attachment to a laminin substrate triggers the tight association with the cytoskeleton of some classes of integrin receptors which are located at the lower cell surface. This association thus immobilizes laminin, and possibly leads to accelerated growth by means of cytoskeletal mechanisms w2x. Further evidence for the association of integrin receptors with cytoskeleton is derived from experiments with chick dorsal root ganglion neurons w73x. Growth cones migrating on laminin were probed with polystyrene beads Ž0.5 mm or 40 nm diameter. conjugated to monoclonal antibodies to b1 integrin and manipulated with laser optical trapping techniques. The beads remained attached to the receptors and served as markers for the surface dynamics of growth cone behavior. The peripheral front of the growth cone, as compared to the base of the growth cone, exhibited increased stable attachment of the larger diameter beads, followed by slow rearward motion. This behav-

Fig. 3. Distribution of b1, a1, and a 3 integrin subunits in cytoskeletal ghosts of PC-12 cells grown on laminin. Confocal analysis was performed after labeling with anti-b1 ŽA,B. or double labeling with anti-a1 ŽC. and anti-a 3 ŽD. antibodies. A shows optical section taken parallel to the substratum and at the level of the lower cell surface. The cytoskeleton retains abundant point contacts composed of b1 integrin subunits. B shows section of the same cell taken perpendicular to the substratum Žarrows indicate positions.. Note the high density of point contacts at the lower cell surface and near absence on the upper surface. C and D, double labeling with anti-a1 ŽC. and anti-a 3 ŽD. antibodies shows that the a1 subunit is retained with the cytoskeleton but a 3 immunoreactivity is lost when ghosts are prepared. Scale bar, 20 mm. Reproduced from Arregui et al. w2x, with the permission of Oxford University Press.

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ior of larger beads linked to integrin receptors at the leading edge of growth was apparently coupled with an actin cytoskeleton that resisted mechanical tether formation when the beads were pulled out from the cell surface. Smaller beads, presumably interacting with smaller aggregates of receptor, were transported preferentially to the growth cone periphery by alternating periods of directed movement and periods of diffusion. Thus, the b1 integrin receptors are organized differently in different regions of the growth cone and exhibit stereotypic patterns of movements during outgrowth on laminin. More integrin receptors at the leading edge of the growth cone may provide a greater transduction of force at the leading edge, as well as a means for steering the growth cone onto favorable substrate w73x. 4.2.2. Non-integrin binding proteins Non-integrin binding proteins to laminin include a peripherally located binding protein Ž Mr s 68,000. that has been characterized in skeletal muscle cell membranes and other cell types, including neurons w56,82,88x. This high affinity binding protein binds to the E1X fragment on a short arm of laminin, specifically to the pentapeptide YIGSR. The 68-kDa binding protein mediates cell attachment, but not neurite outgrowth. Another binding protein Ž Mr s 67,000., initially characterized in fibrosarcoma cells, murine melanoma cells and human breast carcinoma, binds with equal affinity to a hydrophobic sequence LGTIPG in domain V of the b1 chain of laminin Žsee Fig. 1., and to a similar sequence with identical secondary conformation in elastin w57,29x. In addition, the elastin-laminin binding protein acts as a galactolectin, binding to laminin via its polyŽlactosamino. structures. In the presence of lactose, affinity is reduced and ligand-binding protein complexes dissociate. This means that when binding to laminin in domain V of the b1 chain, the ligand can be shed from the cell surface when it interacts with galactosugars w29x. In order to learn more about how this binding protein interacts with the surface of living cells, Mecham and his colleagues w58x exposed fibroblasts to laminin-coated particles of gold and viewed the labeled living cells with a video-enhanced microscope. The laminin-binding protein complexes appeared initially to diffuse randomly in the plane of the membrane and then to move in a directed way toward the rear of the cell, evidently by attaching to actin filaments within the cytoplasm. During the transport period the ligand-binding protein complex was no longer sensitive to lactose and the gold particles were not released from the cell surface, apparently stabilized by interacting with actin filaments. These results are interpreted as illustrating the bidirectional nature of ligand-binding protein interactions, since both the intracellular and extracellular compartments are influenced by the interaction w58x. A laminin-binding protein, cranin Ž Mr s 120,000., which promotes neurite outgrowth, has been purified from

rodent brain and found to bind the E8 fragment in the long arm of the a1 chain w78x. Recently, cranin has been shown to be a form of the dystroglycan receptor w77x. The dystrophin-dystroglycan complex is a laminin-binding protein originally discovered in striated muscle cells where it mediates interaction between laminin in the ECM and actin in the cytoskeleton. Dystroglycan binding to laminin is inhibited by heparin, suggesting that this glycoprotein receptor binds to the end of the long arm of laminin where perlecan Žheparan sulfate proteoglycan. binds ŽFig. 1. w56x. Recent purification of cranin isolated from sheep brain shows that it is a form of dystroglycan that is associated with membranes, and localizes to synapses when rat cerebellum is processed immunocytochemically w77x. The carbohydrates of sheep brain dystroglycanrcranin express high mannoserhybrid N-linked saccharides, terminal Nacetylgalactose amine residues, and the HNK-1 epitope. Brain dystroglycanrcranin has the properties of a mucinlike protein, rather than a proteoglycan and apparently does not need the proteoglycans chondroitin sulfate or heparan sulfate for binding to laminin w77x. A laminin-binding protein Ž120 kDa. related to the dystroglycan complex and to cranin has also been discovered in chick brain w23x. Homology was determined using two-dimensional electrophoresis and protein microsequencing. Characteristics of the 120 kDa binding protein include high affinity binding to the globular ŽG. domain ŽE3. at the end of the long arm where heparin also binds, Ca2q-dependency for binding, and expression in early development of both chick and rat brain w23x. Another binding protein Ž110 kDa. whose interaction with laminin promotes neurite outgrowth, binds specifically to the IKVAV sequence in the a1 chain of the long arm w36,81x. This binding protein has been detected with immunocytochemical methods in neonatal mouse brains where it localizes to bundles of fibers in the thalamus and hippocampus w52x, and in adult rat brains where it localizes to pyramidal neuron cell bodies and processes in the cerebral cortex w32x. Experiments on the role of the laminin binding protein 110 kDa during regeneration in the rat brain show that the binding protein appears in reactive glia and some neuronal processes following either stab wounds or ischemic lesions w32x. Since this laminin-binding protein localizes to normal, non-injured neurons in the brain, as well as to sites of injury in the brain, this implies it may have a dual role w32x. Several lines of evidence have shown that the 110 kDa laminin binding protein behaves like b-amyloid precursor protein Žsee Section 8. w34x. 4.2.3. Lectins and carbohydrate binding Lectins, such as the carbohydrate binding protein 35 ŽCBP-35. that bind galactoside, also bind to laminin via its polylactosamine residues Žsee Section 2. w56,94x. Laminin from different sources may differ in the amount of associated carbohydrate and consequently, in the amount of lectin binding. For example, a comparison of lectin bind-

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ing to laminin from the EHS tumor and from human placenta, using gel overlay and Western blotting techniques, showed preferental binding of galectin-3, a member of the S-lectin family, to EHS laminin w63x. Laminin from EHS tumor also stained more heavily with the periodic acid Schiff reaction than laminin from human placenta, indicating the presence of more carbohydrate residues w63x. Laminin from EHS tumor and from muscle also bind to the lectin Ricinus communis agglutin-1 ŽRCA1. that recognizes terminal galactose residues w90x. Both a1 and b1 chains from EHS tumor bind this lectin, and binding is abolished by simultaneous incubation with Dgalactose. By purifying basement membrane from skeletal muscle, Wadsworth et al. w90x showed another glycoprotein Ž330 kDa. which bound RCA-1 and was disulfidebonded to b1 chains of laminin. The 330 kDa glycoprotein was identical to the a 2 chain of laminin located in the basement membrane associated with extra-synaptic sites in skeletal muscle. They w90x suggest that lectin-binding ability of laminin chains may provide a means of classifying different isoforms and may also reveal functional differences between isoforms that depend on their carbohydrate moieties. Lectin–carbohydrate interactions are significant for laminin-mediated cell behavior. For example, the spreading of B16 mouse melanoma cells on laminin depends on mannoside residues of the putative receptor, the lectin calreticulin w93x. In addition, a glycoprotein cbg 72 Ž Mr s 72,000. that binds concanavalin-A interacts with a laminin substrate on which chick embryo fibroblasts are spreading w59x. Cbg 72 itself and the lectin concanavalin A each prevent chick embryo fibroblasts from spreading on laminin, apparently by competing for sites on laminin. Since cbg 72 resists detergent extraction when the cells are spread on laminin, it appears that it may interact with the cytoskeleton. Another lectin, termed laminin-binding-lectin or LBL, has been detected in basement membranes in muscle, kidney and nerve, and in the connective tissues of the perimysium and perineurium w3x. LBL forms a complex of 190r220 kDa from 70 kDa subunits and binds specifically not only to galactoside residues on laminin, but also to another site distinct from its lectin-binding site. The interpretation that laminin contains two binding sites rests on experiments showing that LBL binding to laminin is not inhibited by galactose w3x. A different binding mechanism between the lactose residues associated with laminin and the cell surface is catalyzed by b1,4 galactosyltransferase ŽGalTase.. This enzyme, located at the surface of lamellipodia and filopodia of growth cones, transfers a galactose residue from a donor UDP-galactose to an acceptor N-acetylglucosamine located on laminin, specifically the E8 fragment of the long arm w7x. GalTase enzyme activity plays a role in neurite outgrowth of PC-12 cells because perturbing enzyme activity using an anti-GalTase antibody reduces initiation of process formation within 30 min to a level that is

9

40% of controls; perturbing enzyme activity with a modifier protein, a-lactalbumin, reduces initiation of process formation within 30 min to a level that is 22% of controls. Likewise, modifying the laminin matrix itself by enzymatically removing the accessible N-acetylglucosamine acceptor sites reduces initiation of process outgrowth within 30 min to a level that is 58% of controls w7x. Continuing elongation of processes over 3–6 h in culture depends less on the GalTase activity than does initiation of outgrowth. Cell adhesion to laminin is not dependent on enzyme activity. Enzyme-substrate binding in this type of interaction could not only bind the cell surface to laminin, but could also release the interactants in the presence of a suitable donor, since binding would be eliminated once the product donor-acceptor complex is generated. Another proposed function for the GalTase system which is not directly involved in binding laminin to cells, is to increase the number of lectin-binding sites on laminin, thereby influencing the kinetics of binding to laminin w56x. A study tracing the effect of lactose on binding of gold-labeled laminin to living cells showed that lactose decreases the affinity of binding to laminin, and does so by decreasing the amount of time laminin is bound Ži.e., increasing the dissociation constant. without affecting the ability of the ligand to bind the cell surface w58x.

5. Mechanism of promotion of neurite outgrowth Laminin affects the velocity and direction of outgrowth by interacting with the growth cone at the growing tip of neurites. The way that laminin functions in this behavior is not clear, but a proposed hypothesis of differential adhesion w43,28x no longer provides a satisfactory explanation. Several lines of evidence have cast doubt on simple adhesivity playing a primary role in laminin’s promotion of growth cone outgrowth. For example, interference reflection microscopy which reveals the degree of association between growth cone and substrate, shows that a preference of growth cones for laminin compared with collagen and fibronectin substrates is not correlated with increased growth cone-substrate association w25x. Further, when growth cones are experimentally detached, they leave behind more substrate-associated membrane, i.e., exhibit a greater degree of contact, on collagen and fibronectin substrates, compared with laminin, even though collagen and fibronectin substrates are poorer promoters of growth w25x. Direct measurements of absolute units of force required to dislodge growth cones show no significant differences among laminin, polylysinerpolyornithine, or plastic substrates, even though growth cone detachment and adhesion clearly are important during the advance of growth cones w97x. Further evidence against differential adhesion as the major mechanism for the function of laminin is that adhesion per se does not determine a growth cones’ preference for a substrate w42x. For example, when given a

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choice between laminin and fibronectin, another glycoprotein of the ECM that is a poorer growth promoter than laminin, growth cones use filopodial scouts to make choices to move onto one or the other substrate ŽFig. 4. w24x. The choice a growth cone makes does not depend on the degree of adhesivity to the substrate, but rather depends on the order of substrates contacted. Filopodial contact leads

to changes in the morphology and behavior of the growth cone, in many cases even before all regions of the growth cone have directly contacted the new substrate ŽFig. 5. w12x. The question of whether the influence of laminin on the extension and growth of processes is ‘permissive’, or ‘instructive’ is difficult to assess, because experiments

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Fig. 5. Time-lapse series of a growth cone initiating growth on a substrate of N-cadherin, a cell adhesion molecule, and contacting laminin at a border region. Images of the growth cone are shown at 0.00 Ža., 8:00 Žb., 10:00 Žc., 14:00 Žd., 15:00 Že., 42:30 Žf., 50:30 Žg., and 59:00 Žh. min progressed time from first frame Ža. Žtime denoted as min:s.. The border is indicated by the dashed line. Note the rapid decrease in growth cone size after first contact with laminin Žc., and the long delay from initial contact until completed cross Žb–h, 51:00 min.. Thick, filopodia-like processes were extended onto the laminin Žd. and retracted multiple times Žnot shown. prior to final cross Žh., as if the growth cone was sampling the new environment. Scale bar, 10 mm. Reproduced from Burden-Gulley, Payne, and Lemmon w12x with permission of Oxford University Press.

vary regarding conditions of the assays such as the source of the neurons, the time in culture until assay, and the presence of adhesive molecules like polyamines that could influence the behavioral outcome w74x. Thus, the influence of laminin on the extension and growth of processes

appears to be ‘permissive’ when rat superior cervical ganglia neurons are grown in tissue culture dishes for 11 h on concentrations of laminin substrate ranging from 0.01– 1.0 mgrcm2 . In this situation quantitative analysis of neurite initiation, outgrowth, and branching shows that the

Fig. 4. Time-lapse sequence of a growth cone on laminin Žabove in each figure. that turned at the border with fibronectin Žbelow in each figure.. A–I: the substratum border is indicated by arrowheads in C–I and is below the field in A and B. Frames are 30 min apart, except for I and H, which are 10 min apart. The field of view has been shifted many times to keep the growth cone in view. A: forward-projecting filopodia extend greater than 40 mm from the distal extent of the growth cone body and contact fibronectin Žnot seen.. B: new filopodia and veil protrude laterally from the growth cone Žarrow., which results in a slight turn to a less direct approach toward the border Žcompare the angle the growth cone makes with respect to the X-axis in A vs. C.. The rate of migration of this growth cone decreased Žsee panel J. at times of apparent increased filopodia sampling of fibronectin in D and G Žarrows., after which the growth cone turned further ŽE,I.. In G Žarrow. a veil extends toward fibronectin, but it is later retracted ŽH.. Scale bar, 10 mm. J: rate of migration of the growth cone in A–I. Letters correspond with the images above. Overall this growth cone underwent only a small decrease in its rate of migration Ž0.54 mmrmin prior to point D, 0.41 mmrmin after point D.. However, for brief periods larger drops in this growth cone’s rate of migration Žarrows. occurred and these rate changes were associated with periods of heightened filopodial sampling of fibronectin ŽD,G.. Reproduced from Gomez and Letourneau w24x with permission of Oxford University Press.

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magnitude of behavioral response does not match the magnitude of change in concentration of the laminin substrate w11x. This leads to the idea that outgrowth is not sensitive to concentration changes in the laminin substrate, and hence that the influence of laminin is ‘permissive’ rather than ‘instructive’. The idea that laminin acts ‘permissively’ by modulating an intrinsic program for neuronal outgrowth is also borne out by evidence from long-term kinetic studies on the development of neuronal polarity w41,51x. Outgrowth of hippocampal neurons in culture is faster on a laminin substrate than on a control substrate of polyamines, but eventually neurons achieve polarity on either substrate. After 12 h in culture on a laminin substrate the major Žlongest. neurite continues to grow whereas minor Žshortest. neurites stop growing, leading to enhancement of neuronal polarity ŽFig. 6. w51x. Once polarized, laminin continues to promote the growth of the longest process,

specifically accelerating the emergence of the axon between 12 and 48 h in culture, thus leading to the differentiated shape of hippocampal neurons that have one long axon and four or five minor processes w41,51x. A detailed examination of the kinetics of laminin-promoted behavior has led to insights about the mechanism of influence for laminin on neurite outgrowth and reveals the importance of both timing and the culture substrate for assessing ‘permissive’ or ‘instructive’ effects on neurite outgrowth. Within 1 h of contact with laminin, NG108-15 cells extend ‘rapid onset’ neurites, thin, highly branched extensions w75x. This early behavior on a plain plastic substrate can be accelerated ten-fold by a ten-fold increase in the concentration of laminin, an apparent ‘instructive’ effect. However, if NG108-15 cells are plated on a laminin substrate on top of polylysine-coated plastic, then a ten-fold increase in laminin concentration results in a much smaller increase in the number of neurites, an apparent ‘permis-

Fig. 6. Time dependence of the effects of substrate-bound laminin and fibronectin on neurite lengths. Cells were maintained for 3, 6, 9, 12, 15, 18, and 21 h in culture and the length of all neurites of process-bearing cells was measured for neurons on the control substrate, poly-D,L-ornithine, ŽC open squares., on laminin ŽLN solid squares., and on fibronectin ŽFN, solid circles.. Results show Ža. the average lengths per cell of all neurites, with significant differences between control and LN or FN substrates except at 15 h, Žb. the average lengths per cell of major neurites, with significant differences between control and LN or FN substrates at all time points; Žc. the average lengths of all minor neurites, with significant differences between control and FN or LN substrates except at 9 h for LN and at 12 h for FN; and Žd. the average length of single minor neurites, with significant differences between control and LN substrate except at 6, 9, 12 h and between control and FN substrate except at 6, 9, 12, 15 h. Reproduced from Lochter and Schachner w57x with permission of Oxford University Press.

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sive’ effect. The interpretation of the latter result is that polylysine inhibits the internalization and recycling of receptor, limiting the maximal rate of neurite extension. This interpretation is supported by studies with polyglutamate and monensin. Polyglutamate partly blocks the nonspecific attachment sites of polylysine and thereby accelerates initial neurite outgrowth on polylysinerlaminin substrates w76x. Monensin inhibits this acceleration by polyglutamate, although monensin alone does not affect initial outgrowth on polylysinerlaminin. The interpretation of these data is that the ionophore monensin equilibrates monovalent cations across the cell surface, and thus inhibits cell-surface receptor internalization. In the presence of polyglutamate, monensin inhibits these maximally stimulated rates of receptor cycling, leading to lessened initial neurite outgrowth w76x. The overall implication is that receptor recycling and internalization are involved in the earliest events of neurite extension mediated by laminin.

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Another early behavioral change mediated by the acute addition of laminin to sympathetic neurons in culture is the acceleration of movement of membranous organelles and microtubules into the growth cones ŽFig. 7. w68x. Furthermore, laminin reduces the retraction of growth cones, leading to the idea that laminin affects the dynamics of growth cone veils and filopodia by lessening the probability that a protrusion will retract. This behavior of the organelles within sympathetic neuronal growth cones is mediated by a specific interaction between laminin and the cell surface because both the organelle invasion and the rate of advance of the growth cone can be inhibited by blocking the b1 subunit of integrin receptor with polyclonal antibody w68x. Further evidence for the importance of the core of organelles within the growth cone is based on the studies of the growth associated protein GAP-43 w1x. Investigators treated chick primary sensory neurons with antisense

Fig. 7. Laminin stimulates filopodial extension and rapid filling of the lamellipodium with membranous organelles advancing from the central region of the growth cone of a sympathetic neuron. a: video-enhanced contrast-differential interference contrast ŽVEC-DIC. micrograph of growth cone after 1 day of growth on polylysine. The central region borders Žarrowhead. on a large, flat lamellipodium with very few visible organelles. Numerous filopodia on the substrate project from the edge of the lamellipodium, and ‘ribs’ extend back toward the central region. The arrow points to a nub of filopodium that was moving toward the rear on the lamellipodium. b: immediately before the addition of laminin Ž35 min after the addition of 25 mgrml ovalbumin.. Ovalbumin did not induce any changes in growth cone morphology; there has been little, if any, advance of the central region or the lamellipodium. c: 12 min after the addition of laminin. The lamellipodium has become engorged with membranous organelles from the central region. Mitochondria and vesicles are visible almost to the edge of the lamellipodium. Several filopodia have lengthened considerably. d: 33 min after the addition of laminin. New protrusions in the lower right have filled with organelles, and a new neurite Žarrowhead. is beginning to form. Bar, 10 mm. Reproduced from Rivas et al. w68x with permission of Cell Press.

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oligonucleotides to deplete the neurons of GAP-43 protein and then compared behavior of the antisense-treated neurons with that of control neurons treated with sense oligonucleotides. The antisense-treated neurons which were depleted of GAP-43 immunoreactivity Žabout 50% of the neurons. exhibited altered growth cone behavior, including poor adhesion to laminin-coated substrates, impaired growth cone spreading, and instability of lamellipodia at the periphery. Further, the GAP-43-depleted growth cones did not exhibit the peripheral accumulation of f-actin filaments typical of actively advancing growth cones. They also did not respond to nerve growth factor-induced spreading, nor to central myelin-derived proteins that induce retraction of growth cones. Aigner and Caroni w1x believe these results mean that GAP-43 is normally involved in the production of the organelle- and microtubule-rich domain that forms the core of the growth cone Žsee w68x. and plays an important role in the stabilization of growth cone structures and the promotion of growth cone adhesion.

6. Laminin in growth cone guidance, pathfinding, and synapse formation 6.1. Guidepost model Several experimental models have been devised to learn more about how laminin might direct neuronal growth cones to follow specific pathways during development of nervous systems. To attempt to mimic pathfinding within the complex environment of the embryonic ECM, one type of experiment used cultures of dorsal root ganglia neurons coupled with cinematographic techniques. Neurons were plated on either laminin or fibronectin substrates and then confronted with ‘guideposts’ in the form of polystyrene beads coated with the alternative substrate ŽFig. 8. w39x. When substrate and guidepost differ with respect to glycoprotein coating, growth cone behavior changes. Only when the carboxy terminal region of laminin is exposed on the guidepost bead does an acceleration in growth cone navigation occur on fibronectin substrate. Furthermore, antibody-pretreatment of laminin-coated guideposts nearly abolishes this acceleration. The interactions between growing tips of neurites and laminin substrates Žor other ECM molecules. are stereotypic and initiated by individual filopodia which thus detect a substrate on a guidepost and direct behavior even before growth cone behavior changes. The nature of the coating on guideposts is irrelevant for filopodial sampling, but a receptor–ligand interaction is required for long-term adhesion to the guidepost. If cells are plated on a fibronectin substrate, a glycoprotein that supports a basal rate of growth cone advance, and confronted with a laminin-coated bead guidepost, they experience a sustained acceleration in growth following filopodial adhesion, dilation of growth cone, and translocation

Fig. 8. Growth cones advancing on fibronectin change their direction upon contacting laminin-model guideposts. A: a series of touches and releases with a single filopodium characterized the initial contact between a growth cone and its new molecular environment, a laminin-model guidepost. B: next, the filopodium established a long-lasting, adhesive contact and started to dilate at its tip. C: then, the growth cone rapidly translocated toward the model guidepost and paused in close proximity. D: finally, the growth cone continued to advance beyond the model guidepost. The dashed line illustrates the predicted straight path of the advancing growth cone in the absence of a laminin-model guidepost. Bead diameter, 4.5 mm. Reproduced from Kuhn et al. w23x with permission of Cell Press.

toward the laminin-coated bead ŽFig. 9. w39x. This acceleration in growth rate is sustained even beyond the guidepost. Conversely, when cells are plated on a laminin substrate and filopodia contacted fibronectin-coated beads, then the cells experience a sustained deceleration in growth to that of fibronectin basal level ŽFig. 10.. Intracellular

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Fig. 9. Only contacts between laminin-model guideposts and growth cones affect the advance of passing growth cones. A,B: transient interactions between laminin-model guideposts and growth cones proper result in a sustained increased rate of advance of passing growth cones. In ŽA., the cumulative distance of a growth cone advancing on fibronectin is plotted against time Žarrow, first filopodial contact to laminin-coated guidepost.. The increase in the slope at t s 34 min, following the pause, is equivalent to a 2.5-fold increased growth rate. Restoration to fibronectin-like levels occurred 15 min later. As shown in ŽB., growth rates were significantly increased during the rapid translocation toward the model guideposts Žasterisk, P - 0.001. and during the immediate 11 min subsequent to the pause Žasterisk, P - 0.001.. Stages of encounter are as follows: I, precontact; II, rapid translocation; III, short-time postcontact; IV, long-time postcontact. Laminin-model guidepost is indicated by dark shading; fibronectin substrate is indicated by light shading. Reproduced from Kuhn et al. w39x with permission of Cell Press.

signaling is required for transduction of guidepost cues, since low concentrations of an inhibitor of protein kinases Žprotein serinerthreonine kinase A and G, and calcium-dependent protein kinase C. reduce the number of growth cones responding even though the filopodial sampling and contacts appear normal. This result suggests that lamininmodel guidepost cues depend on protein kinase-dependent

pathways for transduction of the laminin signal to the cytoskeletal apparatus w39x. 6.2. Patterned substrate model Another type of model system gives neurons in cultures of dorsal root ganglia a choice of substrate by incorporat-

Fig. 10. Fibronectin-model guideposts cause sustained deceleration of growth cones advancing on a laminin substrate. A: the path of one growth cone that encounters a fibronectin-model guidepost Žarrow, initial filopodial contact. is illustrated. Following a 20 min pause, growth cone advance continued at t s 23 min and was significantly decreased, as indicated by the flat slope Ž37 mmrh.. B: growth rates were significantly retarded after transient interactions between growth cones proper and fibronectin-model guideposts Žasterisk, P - 0.001.. Fibronectin-model guidepost, no shading; laminin substrate, light shading. Stages of encounter are as follows: I, precontact; II rapid translocation; III, short time postcontact; IV, long time postcontact. Reproduced from Kuhn et al. w39x with permission of Cell Press.

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L. Luckenbill-Eddsr Brain Research ReÕiews 23 (1997) 1–27

ing patterned substrates on plastic of either laminin alone or laminin alternating with fibronectin. Such experiments attempt to mimic different geometrical patterns of laminin substrates that might occur in the embryonic environment w16,24x. Again, filopodia appear to be the key organelles that initiate changes in growth cone behavior, while integrin receptors on the filopodia play a sensory role in adhesive interactions that activate second messengers that are correlated with changes in growth cone morphology and motility w24x. In order to obtain oriented growth along a track of laminin, filopodia need to detect a border between laminin and the plastic dish. This means that the distance between the laminin track and an adjacent non-adhesive track needs to be greater than the length of the growth cone protrusion. Thus, a growth cone that can span very narrow Ž3 mm. non-adhesive tracks arranged in a multiple parallel pattern does not exhibit oriented growth ŽFig. 11B. and behaves as if on a non-patterned substrate ŽFig. 11A. w16x. On the other hand, growth cones can be guided by single, isolated adhesive tracks that can be as narrow as 2 mm wide. The width of the plane of laminin also influences the polarity of neurons, for on narrow Ž24 mm wide. planes neurite branching was reduced and neurons appear bipolar ŽFig. 11C., whereas on a single, large plane of laminin, neurons appear unoriented ŽFig. 11A.. When growth cones are confronted with a choice between alternating parallel tracks of laminin and fibrinectin substrates, filopodia initiate changes in growth cone behavior even while the growth cone is still on the original substrate w24x, in agreement with results from the model guidepost experiments described above w39x. Dorsal root ganglion neurons appear to be a heterogeneous population with respect to their preference for migrating onto a laminin or a fibronectin substrate when navigating the borderline between the two substrates w24x. This means that patterns of laminin and fibronectin could be instructive and direct axon outgrowth along in vivo pathways where these glycoproteins co-exist.

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6.3. Laminin-3 in synapses

Fig. 12. Neurites seldom grow onto s-laminin-LRE-containing substrates. A: ciliary ganglion neurons were grown overnight on patterned substrates, then viewed under brightfield optics. The patterned substrate was prepared as follows: fusion protein rb2 with the LRE sequence was added to plastic dish, then nickel parallel bar grids were placed in the wells and the dish was irradiated with UV. Area ŽS. shielded by the bars remained active. Then laminin ŽLAM1. was added to the dish, resulting in areas with LAM1 and inactive rb2 ŽL. and areas with LAM1 and active rb2 ŽS.. Neurons grow processes on LAM1, but do not cross the boundary onto active rb2. B: compilation of data from three experiments is shown in which processes crossing onto S-laminin-LRE are compared with processes crossing onto S-laminin-QRE, a synthetic tripeptide that does not have LRE activity. Abscissa is the concentration of fusion protein ŽLRE or QRE. in mgrml. Reproduced from Porter et al. w65x with permission of Cell Press.

Laminin-3 Žs-laminin., an isomer that contains the b2 chain, plays a role in the recognition of synaptic sites during presynaptic differentiation and during regeneration of motor axons, although how this specific localization of the b2 chain arises in unclear w71x. Classic experiments demonstrated that following nerve crush and phagocytic removal of muscle fiber remnants, motor axons regenerated and reinnervated the same synaptic sites, apparently because of a specific signal in the surviving basement

membrane w72x. More recent results with chimeric sequences of b1 and b2 chains transfected into the C2 myoblast cell line have shown that domains I and II near the carboxy-terminal of b2 chain are responsible for the association with other components of the basement membrane within the synapse, and with ‘hot spots’ of acetylcholine receptors on the muscle cell surface during embryonic differentiation w54x. On the other hand, domains V

Fig. 11. The dependence of orientation of chick embryo sensory neurons neurite outgrowth on laminin pattern geometry. A: control, unpatterned laminin shows no orientation. B: 6 mm period pattern Ž3 mm laminin lines and 3 mm spaces. where growth cone can span pattern shows no orientation. C: 24 mm pattern Ž12 mm laminin lines and 12 mm spaces. shows bipolar orientation. Reproduced from Clark et al. w9x with permission of The Company of Biologists Limited.

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and VI of b1 chain promote the association of laminin-1 with extrajunctional components w27,54x. A specific tripeptide ŽLRE. found in three sites within the b2 chain has been implicated as the sequence in b2 to which motorneurons respond during synaptic differentiation, and thus may provide a stop signal during outgrowth which could confine axon terminals to synaptic sites. This has been shown by observing the behavior of ciliary ganglion motor neurons or spinal motor neurons presented with alternating stripes of laminin-1 and LRE as substrates for outgrowth ŽFig. 12A. w65x. Although motor neurons exhibit active growth cones on laminin-1, they stop growing when they encounter the LRE or laminin-3 substrates ŽFig. 12A,B.. The average period of arrest of growth cone activity is 3.8 h. Since the LRE sequence is also found in other components of the synaptic cleft such as agrin, tenascin, and acetylcholinesterase, all of these proteins may provide a signal for motor axon recognition of a synapse during differentiation and regeneration w65x. Laminin-3 also appears in the developing CNS where it is associated with migrating cells, growing axons, and differentiating central capillaries during the development of the blood–brain barrier. Immunocytochemical techniques, confirmed by biochemical methods to detect mRNA, show that the b2 chain is expressed in the subplate of the developing cerebral cortex, a transient region which is involved in migration of neuroblasts and growth of inputs. In the developing spinal cord, specifically in the pial layers associated with the floor plate, the presence of laminin-3 implicates it in the outgrowth of commissural axons w31x.

7. Laminin in developing brain Laminin is found along the routes of migrating neuroblasts w31,45x and growing fiber tracts in the embryonic brain w44,50x and peripheral nervous system w69x. It appears to be functionally important because antibodies to either the b1 subunit of an integrin receptor complex for laminin and fibronectin or to a laminin-heparan sulfate proteoglycan complex produce neural tube defects in chick embryos w46x. In addition, studies of cerebellar granule cell migration in brain slices of postnatal day 8 rat pups using infrared video microscopy show the horizontal and radial translocation of granule cell nuclei within pre-formed processes w47,48x. These results imply an interaction between laminin and the neuronal cytoskeleton that is involved in nuclear movement. Migration is blocked after exposure to an antibody against a neurite outgrowth domain in the g1 chain of laminin w48x. However, neither antibody to intact, native laminin nor pre-immune serum disrupt nuclear translocation of the granule cells. Immunocytochemical methods localize the neurite outgrowth domain of the g1 chain of laminin to areas with Bergmann glial cells whose

fibers have long been implicated in granule cell migration. The role of laminin in cerebellar granule cell migration has also been analyzed in model cell culture systems w22x. When provided with glass fibers coated with various substances on which to migrate, the granule neurons migrate rapidly on laminin-coated fibers using integrin b1 receptors w22x. However, on fibers coated with astroglial cell membranes the granule cells achieve the ‘‘cytology, neuron-fiber apposition and dynamics seen on living glia’’ using the astrotactin receptor w22x. These results illustrate a bit of the complexity of interactions that are involved in granule cell migration. Migration of neurons from the mouse olfactory epithelium appears to be influenced by the E8 domain of laminin, for olfactory neurons which retain their migratory behavior in culture, interact with this domain of laminin using a6b1 integrin receptors w14x. The interaction of migration neurons with laminin is not dependent on adhesive interactions, for laminin is actually ‘anti-adhesive’ in attachment assays. In ‘mapping’ the domain responsible for olfactory neuronal migration, Calof et al. w14x discovered that antibodies to domains ŽE1X or P1X . on the short arms of laminin gave rise to new migration-promoting activity that ‘mapped’ to the distal long arm, some distance from the region where the antibody bound to laminin. The latter activity was mediated by a b1 integrin receptor, but not by a6b1, indicating a new site on E8 was involved. The authors interpret these experiments to mean that antibody binding to P1X induces a change in laminin activity in the long arm ŽE8.. This heightened activity in E8 is normally suppressed by the P1X domain of laminin-1 from EHS tumor. Since laminin-2 Žmerosin. also promotes heightened migratory behavior in olfactory epithelial neurons that is mediated by a b1 integrin, but not a6b1 integrin, the authors suggest that the a 2 chain of laminin-2 may lack the suppressor activity found in P1X domain of laminin-1 w14x. Laminin within the developing nervous system is organized differently from laminin in the adult brain, as suggested by two lines of evidence. First, in embryonic nerve tissues laminin is freely extractable with physiological buffers, indicating that it exists in a soluble, less polymerized or less stable state than in adult basement membranes w21,38x. Second, laminin in embryonic brains occurs in punctate deposits in the ECM, as well as in association with the developing basement membrane sheaths of capillaries, choroid plexus, and the glial limiting membrane ŽFig. 13.. Punctate deposits are associated with a ‘glial’ form of laminin that consists of the b1 and g1 chains only, thus differing from the structure of laminin-1 ŽTable 1. w45,49x. The small, punctate form of laminin in the ECM, which can be demonstrated with immunocytochemical techniques, appears as early as embryonic day ŽE.10 in the rat embryo, well before growing axon bundles invade a particular region ŽFig. 14. w98x. These small punctate deposits, as well as larger punctate deposits which appear

Fig. 13. Small punctiform laminin is distributed in the extracellular matrix of the developing rat brain Ža.. These puncta are not all randomly distributed. They often appear around the blood vessels Žb, arrowheads. in the ependymal cell layer, associate with large cells in the inferior colliculus Žc, arrowheads., or form a unique pattern in the subplate layer of the cortex Žd, arrows.. Scale: a,c, 20 mm; b,d, 50 mm. Courtesy of Dr. F.C. Zhou.

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Fig. 14. Time course of the appearance and intensity of four patterns of laminin in developing brain. The intensity of laminin immunoreactivity represents the descriptive amount of laminin in the brain in each examined stage. Intensity 5 represents the peak amount of laminin during development; 0, none; 1, 2, 3 and 4 proportionately increasing levels. The small punctiform and sheath laminin, known to appear at an earlier stage, rapidly increases in amount at E12–14. Large punctiform and somal laminin appear later at E14–16. The intensity of small punctiform laminin peaks during the stage when active neurites are growing, whereas the intensity of somal laminin peaks at the time when intensity of small punctiform laminin begins to drop. Both small and large punctiform laminin decrease at birth and disappear at early postnatal stages. The somal and sheath laminin persist through adult life at fairly constant levels. Reproduced from Zhou w58x with permission of Elsevier Science Inc.

only in the hippocampus somewhat later in embryonic life, then disappear in the first week or two of postnatal ŽP. life ŽFig. 14.. The punctate deposits are not artifactual because they can be demonstrated on the surfaces of living glial cells whose membranes have not been permeabilized w46x. Besides appearing in extracellular punctate deposits in embryonic stages and early postnatal life, laminin also appears in the soma of neurons beginning around E16 and persists through adult life w32,98x. More than one hypothesis could account for the difference in organization of laminin in embryonic and adult stages. One idea, for example, is that the punctate deposits are the result of differential expression of variant isoforms which do not polymerize into basement membranes. Finding a glial-type isoform with missing a1 chain in the developing brain supports this possibility w46x, although later studies cited above point to the presence of a 2 chain in brain Žsee Section 2.. How the chains in these isoforms are organized is unclear. Future studies using synthetic chains and fragments of various laminin isoforms should prove useful in gaining more information about the organization and role of laminin in the developing brain. Edgar has proposed another idea to explain the distribution of laminin in non-basement membrane ECM, namely that the high levels of expression of laminin subunits

during embryonic stages exceed the rate for polymerization into the basement membrane w21x. This hypothesis predicts that the change in distribution of laminin from embryonic to adult stages involves a down-regulation of laminin gene expression. Evidence for this hypothesis has been obtained from studies of laminin mRNA levels in developing mouse sciatic nerve, which, like developing brain tissue has laminin within the interstitial ECM, rather than exclusively within the basement membrane w38x. Laminin m-RNA levels are highest in early postnatal stages, decrease during the first two postnatal weeks, and reach low adult levels after that w37x. The extractable laminin of embryonic and early postnatal stages that is detected immunocytochemically as punctate deposits in the ECM could more likely express potential cell binding sites than laminin polymerized in basement membranes, and thus could serve as guideposts for neuronal migrations and fiber outgrowth w21x.

8. Laminin and Alzheimer’s disease The fact that laminin is found in lesions within brains of patients with Alzheimer’s Disease ŽAD. and with Down’s syndrome may seem at first glance to involve laminin in

Fig. 15. A: immunocytochemical localization of laminin in the region of a plaque from the hippocampus of a patient with Alzheimer’s disease. The immunostaining with antibodies to whole EHS-laminin-1 is confined to punctate deposits Žarrows. surrounding the core of the plaque, whereas the amyloid-containing core ŽC. is devoid of laminin immunoreactivity. Yellow lipofuscin granules in the plaque exhibit their characteristic autofluorescence Žarrowheads.. B: immunocytochemical localization of laminin in the frontal cortex of a normal human brain shows that laminin immunoreactivity Žarrows. is confined to the basement membranes of blood vessels. Antibody as in panel A. C: immunocytochemical localization of the g1 chain of laminin in the frontal cortex of a brain of a patient with Alzheimer’s disease. Intense immunoreactivity Žarrows. for the g1 chain is seen in astrocytes. Note that the g1 chain-specific antibody does not recognize laminin in basement membranes, only the cellular form of laminin g1. Photos courtesy of Dr. Paivi ¨ Liesi.

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degenerative rather than regenerative or developmental mechanisms. However, the role of laminin in these pathological brains is evidently linked to growth of neuronal processes. Laminin appears in AD brains as punctate deposits associated with vascular basement membranes w64x and with plaques and glia ŽFig. 15. w60x. In senile plaques antibodies either to whole EHS laminin or to epitopes in the P1 fragment from the central cross region of either human or mouse laminin localize to punctate extracellular deposits that surround areas positive for antibody to amyloid b protein ŽFig. 15A.. In normal brains EHS laminin antibody localizes only to basement membrane of blood vessels ŽFig. 15B.. In AD brains, antibodies to the g1 chain localize to glial cells and their fibers, and not to basement membrane ŽFig. 15C. w60x. Further evidence linking laminin with AD shows that when immunoblots and RNA blots of AD brains are compared with control brains, laminin synthesis is increased in AD and the transcript of the g1 chain is 10-times more abundant. Neither AD nor control brains appears to transcribe a1 chain w60x. Binding studies using the enzyme-linked immunosorbent-assay technique ŽELISA. reveal that laminin binds to two sites on the b-amyloid precursor protein ŽAPP. from AD brains, to one site with high affinity and low capacity and to another site with low affinity and high capacity w61x. Free cystein residues are involved in the binding and both binding capacity and affinity are stimulated under reducing conditions. Zn2q also stimulates binding at relatively high concentrations of laminin Žgreater than 1 mgrml., evidently by interacting with a zinc-finger domain of cystein residues in laminin. Further evidence shows that binding may involve ionic interactions because the binding is sensitive to salt. APP also binds to heparan sulfate proteoglycan as well as to laminin and ordered tertiary interactions apparently occur between APP and these ECM components w61x. The interaction between APP and laminin resembles the interaction between the laminin binding protein Ž Mr s 110,000. and the IKVAV sequence from the a1 chain of the long arm of laminin-1 Žsee Section 4.2.2. w34x. Furthermore, antibodies directed either to APP or to 110 kDa laminin binding protein cross react. APP appears to function in neurite outgrowth because when PC-12 cells are transfected with anti-sense messages to APP, the levels of synthesis of both APP and laminin binding protein 110 kDa are decreased and the PC-12 cells’ ability to produce neurites on either laminin or IKVAV is reduced Žsee Section 4.2.2. w34x. These results provide a possible role for APP in both normal and AD or aging brains, one that involves neurite outgrowth, possibly in response to unbalanced proteolytic activity. Evidence for proteolytic activity in senile plaques has been obtained, for example, from immunocytochemical studies of proteoglycans w8x. In addition, the laminin peptide PA22-2, located in the E8 region and containing IKVAV w33x, promotes collagenase IV activity in cell lines when added to the substrate or the

medium of the cells. Since laminin is found in senile plaques, it could also be involved in proteolytic activity there. Taken together, these lines of evidence lead to the following hypothesis: if the IKVAV sequence in the a1 chain of laminin is cryptic in the brain, as preliminary results suggest it is in EHS basement membrane Žw34x; see also w14x., then the unbalanced proteolytic activity typical of AD brain could expose the cryptic sequence IKVAV, leading to abnormal neurite outgrowth, and creating a site for the deposition of APP and amyloid in plaques w34x. Aside from its role in promoting neurite outgrowth in the central and peripheral nervous systems, laminin is involved in the migration and differentiation of neural crest cells into neurons of the enteric nervous system w70x. Using a mouse mutant Ž lsrls . as a model for congenitally aganglionic bowel, Gershon and his colleagues w70x have shown that the presence of more abundant and widespread laminin in the migratory pathway to the bowel appears to inhibit the migration of neural crest cells into the bowel. Thus, these investigators propose that the presence of too much laminin favors neuronal differentiation, rather than the continued migration of cells and the bowel remains aganglionic w70x.

9. Laminin in peripheral regeneration and central nervous system injury 9.1. Peripheral nerÕe regeneration When a nerve is severed or crushed, Wallerian degeneration leads to the removal of axons and myelin sheaths in the distal stump. Schwann cells near the lesion then proliferate and fibroblasts enter the site to bridge the proximal and distal stumps so that regrowing axons and Schwann cells can span the lesion and become re-organized. When axons reach the distal stump, they grow preferentially along the interface between the Schwann cells and the basement membrane w55x. Laminin is correlated in vivo with nerve regeneration because it accumulates at the interface between regenerating axons and the basement membrane tubular scaffold ŽFig. 16. w38x and because its synthesis is strongly up-regulated during peripheral nerve regeneration, along with tenascin and some cell adhesion molecules w55x. When rat sciatic nerve grafts that had been killed by freeze-thawing are spliced into a severed sciatic nerve, pre-treatment of the graft with anti-laminin antibody prevents regenerating axons from recognizing and growing within basement membrane scaffolds associated with the basement membrane tubes w91x. Control, non-antibody treated grafts, as well as anti-fibronectin-treated grafts have 90% or more of the regenerating axons growing inside the basement membrane tube w91x. Laminin has also been used to fill guide-tubes for regenerating axons of severed peripheral nerves of animals. In this situation,

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Fig. 16. Ultrastructural localization of laminin in regenerating adult mouse sciatic nerve. Immunolabelling was performed one week after transection and prior to embedding using polyclonal antibodies against laminin from basement membrane of mouse Engelbreth–Holm–Swarm sarcoma. A–C show the proximal stump of the lesioned nerve. Laminin immunoreactivity is present in basement membranes Žarrows. and associated with surfaces of newly outgrowing axonal sprouts Žarrowheads.. D shows the distal stump of the transected nerve. Immunoreactive material decorates surfaces of axons Žarrowheads. growing along a laminin positive basement membrane tubular scaffold Žarrow.. Collagen fibers are faintly labelled. BM, basement membrane; Ax, axon; Col, collagen. Bars: 0.5 mm. Reproduced from Kuecherer-Ehret et al. w38x with permission of Chapman and Hall.

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either as a component of the basement membrane gel, or in conjunction with other molecules, laminin accelerates regeneration of sciatic nerves in mice w53x. More rapid nerve regeneration means less muscle atrophy and better recovery of function. 9.2. Central nerÕous system The role of laminin in the early events following injury to the mammalian brain remains controversial. The controversy centers on whether the pattern of laminin produced by neurons and astrocytes changes during the early stages of reactive gliosis following injury. Two kinds of experimental paradigms have been studied: one in which a tract like the fornix is lesioned and the histopathological changes in that region are followed using immunocytochemical techniques to detect patterns of laminin compared with control non-injured brains w79x; another in which a tract Že.g., the fornix. is lesioned and the histopathological changes and patterns of laminin in the regions that receive inputs Že.g., the hippocampus. from that tract or give rise to the axons of the tract itself Že.g., the medial septum. are assessed w26x. In the former paradigm of fornix lesions, results show that increased levels of laminin production do not accompany the reactive gliosis stage and the ingrowth of axonal sprouts w79x. In the latter paradigm, results show that the hippocampus whose fornical inputs had been removed displays laminin immunoreactivity in reactive astrocytes. Also in this paradigm, the medial septum which is a source of the severed axons of the fornix contains few laminin-positive cell bodies, compared with non-injured controls. When nerve growth factor ŽNGF. is administered by infusion to the septum at the time of fornix transection, laminin-positive neurons are maintained w26x. The results of this type of experiment point to a role for NGF in the maintenance of central neurons and possibly in the control of laminin production, and illustrate the complexity of trophic influences within the CNS. Both paradigms demonstrate laminin in basement membranes of newly formed blood vessels at the site of transection and in the target hippocampus. Despite the controversy surrounding the role of laminin in regeneration of the mammalian brain, the olfactory bulb of the mammalian brain that exhibits continuous growth, as well as the optic nerves of species like goldfish, and frog that exhibit regeneration all show laminin immunoreactivity associated with astrocytes w46x. This implies that astrocytic laminin is involved with the regenerative potential of these brains.

10. Summary Ž1. Laminin, a large glycoprotein found in basement membranes, in non-basement membrane extracellular matrix ŽECM. in developing stages, and in certain embryonic

and adult neurons, is secreted into the ECM where it interacts with the cell surface, eliciting behaviors such as substrate attachment and process outgrowth. Ž2. A multi-domained molecule consisting of three polypeptide chains, a , b, and g, laminins form a group of heterotrimeric isoforms in various tissues and species. Ž3. Laminin assembly proceeds in two steps, the first of which is the association of the b and g chains, with the critical involvement of the long arm in the triple-stranded coiled-coil structure. Polymerization of laminin results in a self-assembled network which then associates with heparan sulfate proteoglycan and collagen ŽIV. of the basement membrane. Ž4. Several types of cell-surface receptors bind laminin, including members of the integrin family as well as several non-integrin proteins. The carbohydrate moieties of laminin are also involved in cell-surface interactions. The exact means whereby binding of laminin signals a change in cell behavior is uncertain, but probably involves phosphoryation or dephosphorylation of laminin-binding proteins. Ž5. Laminin interacts with the growth cone at the growing tip of neurites to affect the velocity and direction of outgrowth of neurons in culture and within the nervous system. The mechanism whereby laminin exerts its influence is unclear, although differential adhesion to laminin has been shown not to be the key mechanism involved in the promotion of outgrowth of the growth cone. Filopodial sampling is essential, and a lessening of the probability of growth cone retraction appears important. Ž6. Model experiments in culture where growth cones of neurons are presented with two-dimensional substrates that vary with respect the spacing or patterning of laminin and other substrates show that both of these factors influence the rate and directionality of outgrowth. The laminin-3 isoform plays a role in the cessation of outgrowth within the neuromuscular synapse. Ž7. The presence of laminin in the extracellular matrix of the developing central nervous system has been correlated with regions where tracts are growing. In this situation laminin is not only associated with the basement membrane of developing pia and blood vessels, but is also found in small, punctate particles in the extracellular matrix where tract formation occurs. The embryonic laminin appears to be organized differently from that in the adult because it is more readily extracted, compared with adult laminin. Laminin also plays a role in the migration of neural crest cells into the bowel, causing early differentiation of neural crest cells into neurons when too much is present in the pathway of the migrating cells in certain mouse mutants that resemble the human aganglionic colon. Ž8. Laminin is found in lesions of patients with Alzheimer’s disease where it apparently is involved in the growth of neuronal processes associated with senile plaques. Ž9. During peripheral nerve regeneration laminin plays a role in maintaining a scaffold within the basement

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membrane along which regenerating axons grow. Laminin’s role in mammalian central nervous system injury in controversial, although other vertebrates that do exhibit central regeneration have laminin associated with astrocytes in the regenerating regions.

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Acknowledgements I thank Drs. M. Jucker, H.K. Kleinman, and N.R. Smalheiser for critically reading the manuscript, Dr. F.C. Zhou, Department of Anatomy, Indiana University School of Medicine, Indianapolis, IN for providing Fig. 13, and Dr. P. Liesi, Laboratory of Molecular and Cellular Neurobiology, National Institute of Alcohol Abuse and Alcoholism, National Istitutes of Health, Bethesda, MD for providing Fig. 15.

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