Extracellular Matrix Molecules Laminin Research Articles

Ectoderm mesenchymal stem cells promote differentiation and maturation of oligodendrocyte precursor cells

Many neurological diseases are closely associated with demyelination caused by pathological changes of oligodendrocytes. Although intrinsic remyelination occurs after injury, the regeneration efficiency of myelinating oligodendrocytes remains to be improved. Herein, we reported an initiative finding of employing a valuable cell source, namely neural crest-derived ectoderm mesenchymal stem cells (EMSCs), for promoting oligodendrocyte differentiation and maturation by co-culturing oligodendrocyte precursor cells (OPCs) with the EMSCs. The results demonstrated that the OPCs/EMSCs co-culture could remarkably increase the number and length of oligodendrocyte processes in comparison with the mono-cultured OPCs and non-contact OPCs/EMSCs transwell culture. Furthermore, the inhibition experiments revealed that the EMSCs-produced soluble factor Sonic hedgehog, gap junction protein connexin 43 and extracellular matrix molecule laminin accounted for the promoted OPC differentiation since inhibiting the function of anyone of the three proteins led to substantial retraction of processes and detachment of oligodendrocytes. Altogether, OPCs/EMSCs co-culture system could be a paradigmatic approach for promoting differentiation and maturation of oligodendrocytes, and EMSCs will be a promising cell source for the treatment of neurological diseases caused by oligodendrocyte death and demyelination.

LARGE Expression Augments the Glycosylation of Glycoproteins in Addition to α-Dystroglycan Conferring Laminin Binding

Mutations in genes encoding glycosyltransferases (and presumed glycosyltransferases) that affect glycosylation and extracellular matrix binding activity of α-dystroglycan (α-DG) cause congenital muscular dystrophies (CMDs) with central nervous system manifestations. Among the identified genes, LARGE is of particular interest because its overexpression rescues glycosylation defects of α-DG in mutations of not only LARGE but also other CMD-causing genes and restores laminin binding activity of α-DG. It is not known whether LARGE protein glycosylates other proteins in addition to α-DG. In this study, we overexpressed LARGE in DG-deficient cells and analyzed glycosylated proteins by Western blot analysis. Surprisingly, overexpression of LARGE in α-DG-deficient cells led to glycosylation dependent IIH6C4 and VIA4-1 immunoreactivity, despite the prevailing view that these antibodies only recognize glycosylated α-DG. Furthermore, the hyperglycosylated proteins in LARGE-overexpressing cells demonstrated the functional capacity to bind the extracellular matrix molecule laminin and promote laminin assembly at the cell surface, an effect that was blocked by IIH6C4 antibodies. These results indicate that overexpression of LARGE catalyzes the glycosylation of at least one other glycoprotein in addition to α-DG, and that this glycosylation(s) promotes laminin binding activity.

A method for a more complete in vitro Parkinson's model: Slice culture bioassay for modeling maintenance and repair of the nigrostriatal circuit

Slice culture model systems provide a unique opportunity to monitor and lesion brain circuits in a dish. Using a novel approach, we have generated parasagittal slices from mouse brains that preserve, throughout the culture process, the nigrostriatal circuit. These slices can be cultured for approximately 4 weeks with maintenance of normal neuronal cytoarchitecture. Application of the dopamine specific toxin 6-hydroxy dopamine (6-OHDA) induces a significant decline in tyrosine hydroxylase positive cell bodies and fibers. Using a transgenic mouse with green fluorescent protein (GFP) under the control of the tyrosine hydroxylase promoter, we have been able to visualize in real time the loss of GFP expression in the striatum of slices as a result of 6-OHDA exposure. Using these cultures we have demonstrated the feasibility of modeling cellular replacement strategies. GFP-positive embryonic stem cell-derived neuronal precursors can be tracked in real time throughout the experiment and are amenable to patch clamp recording within the slice environment. In addition, cell differentiation can be observed within these slices and the effects of morphogenetic proteins, like the extracellular matrix molecule laminin, drugs or small molecules can be observed. This unique culture system presents a new approach for modeling Parkinson's disease in vitro, and provides a potentially useful new method for screening cell and molecular therapies for neurodegenerative diseases.

The extracellular matrix provides directional cues for neuronal migration during cerebellar development

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Matrix metalloproteinase mediated degradation of basement membrane proteins in Trembler J neuropathy nerves

A single point mutation in peripheral myelin protein 22 (pmp22) of the Trembler-J (TrJ) mouse models the human peripheral neuropathy, Charcot-Marie-Tooth disease type 1 A (CMT1A). An unexplored aspect of this disease is the gradual remodeling of the extracellular matrix in affected nerves. To elucidate the mechanism responsible for these changes, the levels of the extracellular matrix molecules laminin, collagen IV, and fibronectin were determined. In TrJ nerves, laminin is modestly increased while full-length forms of collagen IV and fibronectin are decreased. Matrix metalloproteinases (MMPs) are known to degrade multiple matrix molecules; therefore, nerves were assayed for MMP-2 and MMP-9 proteins. In neuropathy nerves, elevated levels of MMP-2 and MMP-9 were detected on western blots, and gelatin zymography confirmed the up-regulation of gelatinalytic activity in affected samples. Immunostaining studies revealed an increase in the numbers of MMP-2- and MMP-9-expressing cells in TrJ nerves. Cell type-specific immunolabeling showed that infiltrating macrophages are a significant source of both MMP-2 and MMP-9. Finally, the degradation of exogenous collagen IV by TrJ nerve lysates was prevented with a specific MMP inhibitor. Together these observations suggest that infiltration by MMP-expressing macrophages contributes to the remodeling of the TrJ nerve matrix.

Virus one; laminin nil

![][1] High affinity (left), but not low affinity (center), virus can compete with laminin (red). Aclose relative of Lassa fever virus (LFV) may sneak into cells by displacing the extracellular matrix molecule laminin from its normal binding partner, α-dystroglycan, according to

Spatiotemporal patterns of expression of extracellular matrix molecules in the developing and adult rat olfactory system

Using immunocytochemical methods, we have examined extensively the spatial and temporal patterns of expression of three extracellular matrix molecules-laminin, fibronectin, and type IV collagen-in the embryonic, postnatal (days 2 and 11) and adult rat olfactory system. The study started at embryonic day 14 when olfactory fibres and their associated migrating cells course through the nasal mesenchyme. From embryonic day 14 to the adult, a sheet-like pattern of labelling for laminin, fibronectin and type IV collagen was observed along the basal surface of the olfactory epithelium and around the telencephalon. This type of labelling was continuous around the telencephalic vesicle, whereas it appeared disrupted in the basal lamina of the olfactory epithelium to permit exit of the olfactory axons and their associated migrating cells into the mesenchyme. From embryonic day 14 to day 20, punctate labelling for the three molecules studied was observed along the mesenchymal olfactory pathway, the ventral part of the olfactory bulb, the olfactory nerve layer and the presumptive glomerular layer, respectively. By embryonic day 17, the punctate labelling initially detected in the mesenchymal olfactory pathway was replaced by a sheet-like pattern related to the mature basal lamina surrounding the olfactory axon fascicles. Punctate labelling for laminin and type IV collagen persisted in the olfactory nerve layer and around the glomeruli through adult life whereas that of fibronectin declined and disappeared by postnatal day 2. The spatiotemporal distribution of the punctate pattern for laminin, fibronectin and type IV collagen observed in the embryonic olfactory system suggests a role in delineating the pathway for olfactory axon elongation. The continuous expression of laminin and type IV collagen in the adult olfactory bulb may be related to the regenerative activity and high plasticity of the olfactory system.

Expression of extracellular matrix molecules and cell surface molecules in the olfactory nerve pathway during early development

The spatial and temporal expression patterns of several extracellular matrix molecules—laminin and fibronectin and cell surface molecules, neural cell adhesion molecule (NCAM), L1, tenascin, chondroitin sulfate proteoglycan, and peanut agglutinin (PNA) binding sites—were investigated during early olfactory nerve development. NCAM and L1 have similar patterns: They are expressed in the olfactory nerve and on the olfactory receptor neurons (ORNs) commencing with the earliest olfactory axon outgrowth (E12-E15). Their expression patterns suggest that both NCAM and L1 are associated with extension and fasciculation of olfactory axons. A comparison of L1 and olfactory marker protein suggests that L1 is expressed predominantly on immature ORNs. Laminin has an unique punctate staining pattern in the developing olfactory pathway as early as E12. These laminin puncta might play a role in olfactory neurite outgrowth and guidance. At E14, when pioneer olfactory axons enter the brain, the laminin-positive meninges on the surface of the olfactory bulb primordium break down but remain intact in the rest of the telencephalon. This suggests a functional interaction between the olfactory axons and the glial-pial barrier. Fibronectin staining is diffuse throughout the cranial mesenchyme but is absent from the olfactory nerve pathway. No specific patterns of tenascin or chondroitin sulfate, were observed during early olfactory development. PNA binding sites were associated with olfactory axon fasciculation. The expression of several extracellular matrix molecules and cell surface molecules is spatially and temporally regulated in the developing olfactory system. These molecules, thus, may play functional roles in olfactory axon outgrowth, fasciculation, and/or guidance. ©1996 Wiley-Liss, Inc.

Expression and functional roles of neural cell surface molecules and extracellular matrix components during development and regeneration of peripheral nerves

By combining both immunocytochemical and functional investigations, a hypothetical framework will be developed for the molecular mechanisms underlying neuron-glia interactions during development and regeneration of peripheral nerves. In particular, the immunoglobulin-like molecules L1, N-CAM, MAG and P0, the extracellular matrix molecules laminin and tenascin, and the carbohydrates PSA and L2/HNK-1 will be considered. During early stages of limb bud innervation in embryos, L1 and N-CAM are expressed on axons and Schwann cells and are involved in axonal fasciculation, whereas tenascin is thought to be involved in forming a scaffold around the nerve possibly preventing axons and/or Schwann cells from leaving the nerve. PSA has been shown to be involved in pathway selection at initial stages of limb bud innervation. Later on, when motor axons enter muscles, the carbohydrates determine the branching pattern of the nerves. During myelination, L1 appears to play a pivotal role during the formation of the first Schwann cell loops around the prospective myelin-containing axons. MAG and P0 appear also to be functionally involved at initial stages of myelin formation. Additionally, MAG may contribute to the formation and maintenance of non-compacted myelin and axon-Schwann cell apposition whereas P0 is involved in myelin compaction. Under regenerative conditions, L1, N-CAM, laminin, and tenascin are strongly up-regulated by denervated Schwann cells. In vitro observations strongly suggest that these molecules might foster axonal regeneration. The carbohydrate PSA is confined to regrowing axons and is also a candidate to support axonal regrowth. L2/HNK-1, which is found on motor axon-associated Schwann cells, may provide regenerating motor axons with a selective advantage over others resulting in appropriate reinnervation of motor pathways. Since many of the functional studies this review refers to have been performed in vitro, some of the conclusions drawn need reexamination in vivo. Gene manipulations, such as the generation of null mutants followed by a thorough morphological and immunocytochemical investigation may be a powerful tool to resolve this problem.

Extracellular matrix molecules in development and regeneration of the leech CNS

As neurons grow to their targets their processes elongate, branch and form specialized endings into which are inserted appropriate ion channels. Our aim has been to analyse the role of the extracellular matrix molecules laminin and tenascin in inducing growth and in determining the form and physiological properties of growing neurites. A preparation in which development and regeneration can be followed at the cellular and molecular level in the animal and in tissue culture is the central nervous system (CNS) of the leech. In leech extracellular matrix (ECM) both laminin and tenascin are present; the molecules are structurally similar but not identical to their vertebrate counterparts. Tenascin extracted from leech ECM shows a typical hexabrachial structure whereas laminin shows a typical cruciform structure in rotary shadowed preparations. Leech laminin purified by means of a monoclonal antibody is a molecule of about 1000 kDa, with a polypeptide composition of 340, 200, 180 and 160 kDa. Substrates that contain tenascin or laminin produce rapid and reliable outgrowth of neurites by identified cells. A remarkable finding is that the outgrowth pattern produced by an individual neuron depends in part on its identity, in part on the substrate upon which it is placed. For example, a Retzius cell grows in a quite different configuration and far more rapidly on laminin substrate than does another type of neuron containing the same transmitter (serotonin); and the pattern of outgrowth of the Retzius cell is different on laminin and on the plant lectin Con A (concanavalin A). Thus Con A induces the growth of processes that are shorter, thicker, more curved and contain fewer calcium channels than those grown on laminin. To determine whether laminin can also influence neurite outgrowth in the animal, immunocytological techniques have been used to follow its distribution in the extracellular matrix of normal, developing and regenerating leech CNS. In adult leeches neuronal processes in the CNS are not in contact with laminin which is confined to the surrounding extracellular matrix. In embryos however, laminin staining appears between ganglionic primordia along the pathways that neurons will follow. Similarly, after injury to the adult CNS, laminin accumulates at the very sites at which sprouting and regeneration begin. How the laminin becomes redistributed to appear in the region of injury has not yet been established. Together these findings suggest a key role for laminin and for other extracellular matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate interactions affecting motor growth cone guidance during development and regeneration.

Most serious injuries of spinal nerves or roots in man and other higher vertebrates lead to permanent loss of control of skeletal muscles. In some cases this may be due to a failure of motor axons to regenerate, although even when functional neuromuscular connections are re-established, coordinated use of body and limb muscles may be absent. In both mammals and lower vertebrates, damaged motor axons usually regrow and reform functional connections with muscles, although these connections are often inappropriate. The selectivity of reinnervation is improved by maintaining alignment of the severed ends of the nerve. Thus, factors operating near the lesion site may direct regenerating motor axons into fascicles in the distal nerve stump that lead to inappropriate muscles. The identity of some of these factors is suggested by recent studies of developing systems which have shown that motor axons are directed in their growth. (a) The filopodia of their growth cones sample a limited region of the periphery. If motor growth cones extend too far from their normal pathways they establish connections with inappropriate muscles. (b) Motor growth cones normally extend into regions of embryos rich in the extracellular matrix molecule laminin, and avoid regions containing fibronectin. Moreover, motor growth cones extend on laminin but not on fibronectin substrates in vitro. In peripheral nerves, these two molecules are differentially distributed; laminin is expressed by Schwann cells in the endoneurium whereas fibronectin is expressed by fibroblasts primarily in the perineurium. These studies suggest that regenerating motor growth cones may be directed to appropriate muscles if their original fascicles within the distal nerve stump are within filopodial reach but may not be able to escape the fibronectin-rich perineurial sheath once directed into an inappropriate fascicle.

Neural crest cell migratory pathways in the trunk of the chick embryo

Neural crest cells migrate during embryogenesis to give rise to segmented structures of the vertebrate peripheral nervous system: namely, the dorsal root ganglia and the sympathetic chain. However, neural crest cells arise from the dorsal neural tube where they are apparently unsegmented. It is generally agreed that the somites impose segmentation on migrating crest cells, but there is a disagreement about two basic questions: (1) exactly what pathways do neural crest cells use to move through or around somites, and (2) do neural crest cells actively migrate or are they passively dispersed by the movement of somite cells? The answers to both questions are critically important to any further understanding of the mechanisms underlying the precise distribution of the neural crest cells that develop into ganglia. We have done an exhaustive study of the locations of neural crest cells in chick embryos during early stages of their movement, using antibodies to neural crest cells (HNK-1), to neural filament-associated protein in growing nerve processes ( E C8 ), and to the extracellular matrix molecule laminin. Our results show that (1) Some neural crest cells invade the extracellular space between adjacent somites, but the apparent majority move into the somites themselves along the border between the dermatome/myotome (DM) and the sclerotome. (2) Neural crest cells remain closely associated with the anterior half of the DM of developing somites as they travel, suggesting that the basal lamina of the DM may be used as a migratory substratum. Supporting this idea is our observation that the development of the DM basal lamina coincides in time and location with the onset of crest migration through the somite. (3) The leading front of neural crest cells advance through the somite while the length of the DM pathway remains constant, suggesting active locomotion, at least in this early phase of development. (4) Neural crest cells leave the DM at a later stage of development to associate with the dorsal aorta, where sympathetic ganglia form, and to associate with newly emerging fibers of the ventral root nerve, where they presumably give rise to neuronal supportive cells. Thus we propose that the establishment of the segmental pattern of the peripheral ganglia and nerves depends on the timely development of appropriate substrata to guide and distribute migrating neural crest cells during the early stages of embryogenesis.