摘要:

AbstractIn a model of astrogliosisin vitro, cultured cortical astrocytes were triggered into a functionally reactive state by an immobilized fragment of the β‐amyloid peptide. Induced astrocytes produced an extracellular matrix that inhibited the outgrowth of embryonic CNS axons. Within the extracellular matrix deposited by reactive astrocytes, we found an overall increase in the deposition of chondroitin sulphate that accounted for the inhibition. Specifically, we have detected an increased biosynthesis of a small chondroitin/heparan sulphate proteoglycan that is a potent inhibitor of axon outgrowth. We further suggest that this proteoglycan, or related molecules yet to be discovered, may play a role in gliosis‐mediated regenerative failure of CNS axons.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00004-4

出版商:Wiley    

年代:1999

数据来源: WILEY

摘要:

AbstractAstrocytes appear to be excluded from blood vessels. In order to test this observation, pial blood vessels were seeded upon cultures of purified E14 fetal astrocytes. One to seven days later co‐cultures were immunohistochemically stained for laminin and fibronectin. Confocal microscopy revealed that laminin‐positive cultured astrocytes migrated from the coverslip to and on to the surface of the blood vessels. The fetal astrocytes migrated along the basement membrane but did not enter the blood vessel. Viable (live) basement membrane is a barrier to astrocyte intravasation and is a pathway for astrocyte migration.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00005-6

出版商:Wiley    

年代:1999

数据来源: WILEY

摘要:

AbstractIntegrins are cell surface receptors for a variety of extracellular matrix molecules including fibronectin, laminin and collagens. Although their role in development is not completely understood, they are likely to have important functions in cell migration and axon guidance. To characterize the types of integrins expressed in the developing nervous system, we have used monoclonal antibodies against α7‐ and αv‐integrin subunits to examine the distribution of these subunits in the early chick embryo. Low levels of α7immunoreactivity were first observed in the neural tube and developing myotome of stage 17 embryos (E2.5). Although low levels of α7expression were associated with most neuroepithelial cells, distinct α7immunoreactivity was first detected in the ventrolateral portions of the neural tube at a stage corresponding to the time when the first neurons differentiate. Its distribution pattern overlapped with that of commissural neurons in the developing spinal cord. α7was also prominently localized to the motor neurons and their axons emanating from the neural tube. In addition, α7immunoreactivity was observed on a subpopulation of trunk neural crest cells migrating through the somitic sclerotome. At later stages, α7expression was observed in other nervous system structures such as the pigmented retinal epithelial cells. In addition to its distribution in the developing nervous system, α7immunoreactivity was associated with early myotomal cells shortly after myotome formation and its expression persisted throughout myotome development. In contrast to α7, αv‐integrin had a limited distribution in the nervous system, being expressed only at low levels in the neural tube. However, αvdisplayed prominent immunoreactivity in the myotome and in endothelial cells of the dorsal aorta. The results suggest that α7‐integrin is one of the prevalent integrin subunits on neurons and axons in the developing spinal cord.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00006-8

出版商:Wiley    

年代:1999

数据来源: WILEY

摘要:

AbstractWe have established the presence of at least two large chondroitin sulfate proteoglycans in the developing chick brain, one that reacts exclusively with HNK‐1, a carbohydrate epitope found on several neural specific molecules, and one that reacts with S103L, a defined peptide epitope in the CS‐2 domain of the cartilage‐specific chondroitin sulfate proteoglycan (CSPG), aggrecan. In order to determine the relationships between the two distinct S103L‐reactive CSPGs from cartilage (chondrocytes) and brain (neurons), as well as among the three large CSPGs expressed in brain, S103L, HNK‐1 and versican, we studied the expression of these multiple proteoglycan species in the brain of nanomelic chicks. We have previously shown that homozygous embryos expressing the nanomelic phenotype exhibit a single point mutation in the aggrecan gene. In the present study, the S103L CSPG is not accumulated or synthesized by embryonic chick CNS tissue or E8CH neuronal cultures derived from nanomelic chick embryo cerebral hemispheres. In contrast, expression of both versican and the HNK‐1 CSPG was normal in the mutant embryo CNS. Pulse chase experiments demonstrated the presence of the 380 kDa precursor in normal neurons and the 300 kDa truncated precursor in nanomelic neurons. Northern blot analysis revealed normal‐sized mRNA but reduced levels of expression of the S103L CSPG message in nanomelic neurons, while expression of the versican message was comparable in normal and nanomelic neurons. Most conclusively, the point mutation previously identified in nanomelic cartilage mRNA was also identified in nanomelic brain mRNA. Together these results provide evidence that a single aggrecan gene is expressed in both cartilage and CNS tissue leading to the production of identical core proteins which then undergo differential and tissue‐specific post‐translation processing, resulting in the characteristic tissue‐specific proteoglycans. Furthermore, versican and the HNK‐1 CSPG, although structurally and chemically similar to the S103L CSPG, are the products of separate genes.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00007-X

出版商:Wiley    

年代:1999

数据来源: WILEY

摘要:

AbstractThe growth patterns of axons and dendrites differ with respect to their number, length, branching, and spatial orientation; therefore, it is likely that these processes differ in their growth requirements. To examine this hypothesis, we have been analyzing the responses of cultured rat sympathetic neurons to three types of stimuli: large structural proteins of the extracellular matrix, matrix‐associated growth factors, and neurotrophins. Purified structural proteins such as laminin and collagen IV have been found to promote only axonal growth; whereas the matrix associated growth factor, osteogenic protein‐1, selectively stimulates dendritic growth. In contrast, nerve growth factor modulates the growth of both types of processes. These data suggest that process‐specific interactions with the extracellular environment may be critical determinants of cell shape in neurons. Perinatal rat sympathetic neurons grown in culture in the absence of serum or glial cells extend a single process which is axonal in nature. Exposure to osteogenic protein‐1 causes the formation of additional processes which express the morphological, cytoskeletal, and ultrastructural characteristics of dendrites. Consistent with observations on the regulation of dendritic growth in sympathetic neuronsin situ, the dendrite‐promoting activity of osteogenic protein‐1 is independent of synaptic or electrical activity, but is modulated by nerve growth factor. In the presence of optimal concentrations of osteogenic protein‐1 and nerve growth factor, the size of the dendritic arbor extended by cultured sympathetic neurons approximates that seenin situat comparable developmental stages. Osteogenic protein‐1 does not promote dendritic growth in cultured neurons obtained from embryonic ciliary, dorsal root, trigeminal or nodose ganglia, suggesting that its morphogenetic effects are cell selective. Since mRNA for osteogenic protein‐1 is expressed in mature as well as embryonic target tissues of the sympathetic nervous system, we also examined the effects of osteogenic protein‐1 on cultures of sympathetic neurons derived from adult rats. Consistent with results obtained with perinatal neurons, osteogenic protein‐1 selectively promoted dendritic growth in adult neurons. These data suggest that this matrix‐associated growth factor could play a role not only in the morphogenesis of the developing nervous system, but also in the maintenance and remodeling of dendritic structures in the mature animal.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00008-1

出版商:Wiley    

年代:1999

数据来源: WILEY

摘要:

AbstractTenascin‐R (TN‐R) is an extracellular matrix protein associated with the surface of neurons and glial cells. Immunohistological studies reveal that TN‐R shows a restricted expression pattern in the developing nervous system. TN‐R is the smallest member of the tenascin family and is composed of four structural motifs: a cysteine‐rich segment at the N‐terminus is followed by 4.5 EGF‐like repeats. This region is followed by 9 consecutive fibronectin type III (FNIII)‐like domains and at the C‐terminus TN‐R is related to the β‐ and γ‐ chains of fibrinogen. TN‐R forms oligomeric structures as revealed by rotary shadowing electron microscopy of immunoaffinity‐purified TN‐R. TN‐R interacts with the axon‐associated F11 protein which results in an enhancement of F11 mediated neurite extension inin vitroassays. In short‐term adhesion assays it was found that neural but not fibroblastic cells attach effectively on immobilized TN‐R. The cell attachment site within TN‐R was allocated to FNIII domain 8 while the site interacting with the F11 protein could be mapped to FNIII domain 2–3.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00009-3

出版商:Wiley    

年代:1999

数据来源: WILEY

摘要:

AbstractChondroitin sulfate proteoglycans, cell surface and extracellular matrix molecules in both neural and non‐neural tissues, are highly regulated during normal development. Entire proteoglycan molecules may be either up‐regulated or down‐regulated, or only the chondroitin sulfate glycosaminoglycan portions of these molecules may be modified. Subtle changes in the chemistries of chondroitin sulfate chains can now be identified through the use of a panel of anti‐chondroitin sulfate monoclonal antibodies. Each of these antibodies recognizes specific chemical structures which are non‐randomly dispersed along the lengths of chondroitin sulfate chains. The location of individual epitopes within defined domains in these chains is demonstrated through controlled treatments of aggrecan with chondroitinase ABC, whereby portions of these chains are removed from the non‐reducing terminal ends and where the remainder of the chains remains covalently attached to the core protein. In these situations, some epitopes, such as those recognized by antibodies CS‐56 and 6C3, can be removed without loss of other epitopes, such as that recognized by antibody 4C3. The independent expression of individual epitopes is demonstrated by immunocytochemical analyses of developing skin appendages in embryonic chicks and fetal humans. These are sites where highly patterned morphogenetic movements result from epithelial‐mesenchymal interactions. In both chicks and humans, some epitopes are constitutively expressed while others are strictly regulated in the mesenchymal portions of the developing skin appendages. These data strongly suggest that chondroitin sulfate proteoglycans, including their chondroitin sulfate chains, have important roles in regulating these epithelial—mesenchymal interactions. Furthermore, these data underscore the significance of the aforementioned observation that individual epitopes are located in specific domains within chondroitin sulfate chains. The highly organized expression of chondroitin sulfate proteoglycans in the development of the central nervous system strongly argues for a similar role for these molecules in the organs that comprise this system.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00010-X

出版商:Wiley    

年代:1999

数据来源: WILEY

摘要:

AbstractProteoglycans involved in the shaping of the developing brain are often preserved in the adult brain in more restricted locations. We have studied the fate of DSD‐1‐PG, a chondroitin sulfate proteoglycan containing the hybrid epitope DSD‐1. DSD‐1‐PG exerts neurite outgrowth promoting activity and has been shown to occur in the developing brain during late brain development and into adulthood. In the adult rat brain, monoclonal and polyclonal antibodies against DSD‐1‐PG labelled only the circumference of a selected subpopulation of neurons. These nerve cells invariably expressed the calcium‐binding protein parvalbumin. The label occupied the extracellular space in close vicinity to the cell body, surrounding axon terminals and glial end feet, but was absent from synaptic clefts. DSD‐1‐PG is thus shown to be an additional representative of the growing list of substances found in perineuronal locations in the adult mammalian brain.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00011-1

出版商:Wiley    

年代:1999

数据来源: WILEY

摘要:

AbstractThe up‐regulation of the keratan sulfate proteoglycan (ABAKAN) was examined using indirect immunohistochemical methods. Previous studies indicate that the keratan sulfate proteoglycan is associated with astrocytes in the optic nerve and in the developing rat brain. In model culture systems, this proteoglycan is capable of inhibiting the growth of neurites over laminin. To determine whether the proteoglycan is up‐regulated specifically during reactive gliosis, stab wounds were made in the cerebral cortex of early postnatal rats, and the up‐regulation of the proteoglycan was related to the developmentally regulated gliotic response to injury. Following a stab wound in the cortex of the late postnatal rat, reactive gliosis was consistently observed along with an up‐regulation of ABAKAN. When the cortex was injured on postnatal day 2, there was a variable gliotic response and considerable variation in the regulation of proteoglycan expression. Biochemical analysis revealed that ABAKAN is a large proteoglycan with multiple keratan sulfate side‐chains, at least one chondroitin sulfate side‐chain and at least one additional carbohydrate chain with a terminal 3‐sulfoglucuronic acid. Taken together, these data demonstrate that the boundary proteoglycan ABAKAN is also associated with reactive gliosis during early postnatal development.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00012-3

出版商:Wiley    

年代:1999

数据来源: WILEY

摘要:

AbstractIntegrin α6β1 is a laminin receptor involved in adhesion and neurite extension of retinal neurons on laminin. The present study was carried out to understand some of the intracellular mechanisms which allow integrin‐mediated neurite extension on laminin in primary neuronal cultures. Both integrin‐mediated adhesion to laminin and antibody‐induced integrin clustering resulted in the increased tyrosine phosphorylation of a 120 kDa polypeptide which was identified as the focal adhesion kinase. The kinetics of phosphorylation and dephopsphorylation of this kinase were dramatically different in neurons plated on laminin, than in neurons in which the receptors were clustered with anti‐integrin antibodies. To look at possible interactions of the focal adhesion kinase with integrins, we made use of sucrose velocity gradients, which have allowed the identification of a large complex containing the α6β1 laminin receptor. Analysis of the gradients showed that the focal adhesion kinase was not associated with the integrin receptors under these experimental conditions, while about 26% of the c‐Src kinase codistributed with the integrin receptor complex, and showed a molecular size and a distribution similar to that of a 59 kDa phosphoprotein co‐migrating with the α6β1 receptor. Our results suggest that integrin‐induced tyrosine phosphorylation is an early intracellular event during neuronal adhesion, and that the integrin‐mediated increase in tyrosine phosphorylation of the focal adhesion kinase is not sufficient per se for the induction of neurite outgrowth. Furthermore, our data indicate that Src kinase may be involved in integrin‐mediated neuronal interactions with laminin.

ISSN:0736-5748    

DOI:10.1016/0736-5748(96)00013-5

出版商:Wiley    

年代:1999

数据来源: WILEY