Bioactive surface coatings for nanoscale instruments: effects on CNS neurons.

A method is described for depositing onto medical instruments highly biocompatible and bioactive surface coatings that can promote and stabilize cell attachment. The coatings were made by first depositing thin films of materials, such as diamond-like carbon, or metals, including tantalum, tungsten, platinum, gold, iridium, palladium, and brass. These surfaces were further altered to either promote or inhibit cell growth and spreading by an additional overcoat of biological materials, including the extracellular matrix proteins, laminin, fibronectin, and collagen IV. The deposition technique used a metal or carbon plasma, and the important properties of film adhesion, hardness, density, and smoothness are tailored by control of the ion bombardment energy. The films are translucent enough to permit high resolution light microscopy for rapid and detailed examination of tissue response. These bioactive substrates have been tested on primary central nervous system neurons, and the growth response is excellent. Equally successful have been our attempts to anchor neurons, without associated proliferation of non-neuronal cells, using coatings of poly-d-lysine. The method and the materials could have important ramifications in a number of areas of research and biotechnology, for example for chronic implantation of microelectrode arrays in the cerebral cortex for neuroprosthetic and neural monitoring application and for research on the human central nervous system. Possible application in nonneuronal fields, such as for coronary artery stents and pacemaker electrodes, also are discussed.

Agrin inhibits neurite outgrowth but promotes attachment of embryonic motor and sensory neurons.

Agrin is a secreted glycoprotein with the ability to cluster cell surface molecules, including the nicotinic acetylcholine receptor (AchR) on muscle cells. Alternate splicing of agrin mRNA results in a family of agrin proteins which differ in their clustering potency. Neuronal-specific isoforms with the highest clustering activity play a role in clustering postsynaptic proteins at the neuromuscular junction. However, the function of agrin isoforms expressed in many nonneuronal tissues, and only weakly active in clustering assays, remains obscure. Monolayer cultures of Chinese hamster ovary (CHO) cells expressing a neuronal (agrin-19) or a nonneuronal (agrin-0) form of agrin were used to assay the effect of agrin on neurite outgrowth and cell attachment. These results were compared to outgrowth on control CHO cells expressing only drug resistance and on regions of CHO-agrin monolayers not expressing detectable levels of agrin. Neurite extension on confluent monolayers of agrin-0- or -19-expressing CHO cells was reduced substantially below that of controls. In one experiment neurite lengths were compared at 2 and 3 days after plating and suggested that neurite outgrowth may be stopped and not simply retarded. Attachment of sensory or motoneurons was nearly twofold higher to agrin monolayers than to control cells, showing that the inhibition is not a result of a nonpermissive environment. An agrin construct missing the C-terminal half, removing the major site of variability and clustering activity, was also tested. This construct did not reduce outgrowth, suggesting that the C-terminal half of the protein may be important in stopping growth as well as inducing clustering. These results expand the role of agrin in synaptogenesis as it may provide a stop signal at the myofiber surface and may anchor the presynaptic fibers to the eventual motor endplate .

Functional partitioning of beta1 integrins revealed by activating and inhibitory mAbs.

Integrins exist in different activation states on the surfaces of cells. Addition of the proper signal, ligand, or antibody can alter the activation state of these molecules. We report here the identification of two immunocytochemically distinct populations of beta1 integrins on fixed embryonic chick dermal fibroblasts. One population, recognized by the integrin activating mAb TASC, localizes to discrete regions of the cell, most likely focal contacts. These integrins co-localize with other proteins, such as vinculin and F-actin, and their retention at these sites is dependent on the actin cytoskeleton. The other population, identified with the inhibitory mAb W1B10, is more evenly distributed throughout the cell surface, and its pattern remains unchanged after disruption of the actin cytoskeleton. Double labeling experiments using Fab fragments of TASC alongside whole W1B10 IgG revealed non-overlapping staining patterns. These results show that it is possible to visualize and study discrete populations of integrins on cell surfaces using two different antibodies. We hypothesize that these antibodies report differences in the distribution of receptors in two different states. A model is proposed describing the ligand independent recruitment of integrins based on these findings and results from other labs.

Heterologous expression of alpha 1-integrin cDNA generates variable ligand specificities and alterations in cell shape.

Integrins can mediate a diverse variety of functions that are regulated by unknown mechanisms. Integrin alpha 1 beta 1 can serve as a receptor for laminin-1 and collagen in certain cell types, but is a receptor for only collagen in others. To examine the molecular basis of this difference in specificity, three cell types were transfected with cDNA for the rat alpha 1 subunit. Following transfection with rat alpha 1, pluripotential hematopoietic human K562 cells exhibited alpha 1 beta 1-dependent attachment to collagen IV, but not laminin-1, unless activating antibody TS2/16 was added. The attachment to collagen IV stimulated the elaboration of a spread morphology resembling a differentiated megakarocyte with extensive processes which were absent in response to all other substrates. When MRC-5 cells, a human fibroblastic cell, or RD cells, a human rhabdomyosarcoma line, were transfected with the identical alpha 1-integrin construct, rat alpha 1 beta 1-dependent attachment to both collagen IV and laminin-1 was seen. Therefore differences in ligand specificity can be generated by translation of an identical integrin alpha 1 beta 1 mRNA in different cell types. Despite differences in ligand binding, alpha 1 cDNA-transfected K562 and RD cells express an alpha 1 subunit that appears to be antigenically and electrophoretically similar. Small differences in glycosylation were apparent, and correlated with changes in ligand specificity. Together these results show for the first time that identical cDNAs, absent activating antibodies or other manipulations, can change ligand selectivity and better establish the importance of cellular context in determining integrin function. Moreover they show that select integrins can shift the differentiated state of pluripotential cells.

Neuron-specific expression of high-molecular-weight clathrin light chain.

High-molecular-weight forms of clathrin light chains LCa and LCb contain inserted sequences and are expressed in brain tissue but have not been observed in peripheral tissues. Monoclonal antibodies specific for the high-molecular-weight form of LCb and all forms of LCa were used to analyze their expression in different species and different neuronal cell types. High-molecular-weight light chains were found in bovine, rat, mouse, chicken, and human brain, indicating a conserved pattern of expression. Neuron-specific expression of the high-molecular-weight light chains was suggested by analysis of human brain gray matter and white matter. The former contained a higher proportion of light chains with insertion sequences. Immunohistochemical analysis localized the high-molecular-weight form of LCb to synapses and neuronal perikarya, but not to glial cells. Immunofluorescent labeling of cultured chicken dorsal root ganglia confirmed expression in neurons but not Schwann cells. These results indicate that the high-molecular-weight forms of clathrin light chains are restricted in expression and found in neuronal cells.

Molecular cloning of the rat integrin alpha 1-subunit: a receptor for laminin and collagen.

Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.

A role for apolipoprotein E, apolipoprotein A-I, and low density lipoprotein receptors in cholesterol transport during regeneration and remyelination of the rat sciatic nerve.

Recent work has demonstrated that apo E secretion and accumulation increase in the regenerating peripheral nerve. The fact that apoE, in conjunction with apoA-I and LDL receptors, participates in a well-established lipid transfer system raised the possibility that apoE is also involved in lipid transport in the injured nerve. In the present study of the crushed rat sciatic nerve, a combination of techniques was used to trace the cellular associations of apoE, apoA-I, and the LDL receptor during nerve repair and to determine the distribution of lipid at each stage. After a crush injury, as axons died and Schwann cells reabsorbed myelin, resident and monocyte-derived macrophages produced large quantities of apoE distal to the injury site. As axons regenerated in the first week, their tips contained a high concentration of LDL receptors. After axon regeneration, apoE and apoA-I began to accumulate distal to the injury site and macrophages became increasingly cholesterol-loaded. As remyelination began in the second and third weeks after injury, Schwann cells exhausted their cholesterol stores, then displayed increased LDL receptors. Depletion of macrophage cholesterol stores followed over the next several weeks. During this stage of regeneration, apoE and apoA-I were present in the extracellular matrix as components of cholesterol-rich lipoproteins. Our results demonstrate that the regenerating peripheral nerve possesses the components of a cholesterol transfer mechanism, and the sequence of events suggests that this mechanism supplies the cholesterol required for rapid membrane biogenesis during axon regeneration and remyelination.

Identification of a neuronal laminin receptor: an Mr 200K/120K integrin heterodimer that binds laminin in a divalent cation-dependent manner.

Neuronal interactions with extracellular matrix (ECM) components are crucial for axon growth and guidance during development and nerve regeneration. Laminin (LN), a prominent ECM glycoprotein, promotes neuronal survival and axon growth. To identify neuronal receptors for LN, we looked for cell surface proteins on the neuronal cell line B50 that bind LN. An integrin alpha/beta 1 dimeric receptor was identified and purified using lectin and LN affinity chromatography. The purified integrin contains two subunits with Mrs of 200 K and 120 K that bind LN specifically in the presence, but not the absence, of divalent cations (Ca2+/Mg2+ or Ca2+/Mn2+). The Mr 120 K protein was identified as the rat integrin beta 1 subunit using two beta 1 subunit-specific antibodies, and was shown to form a noncovalent complex with the Mr 200K putative alpha subunit. Since neurons and neuronal cell lines express similar integrin beta 1-class heterodimers that mediate attachment and process outgrowth on LN, the Mr 200K/120K complex identified here is likely to be an important laminin receptor used by neurons. This integrin may also mediate binding to LN by many nonneuronal cell types.

Examination of a nerve injury-induced, 37 kDa protein: purification and characterization.

Following traumatic injury to the adult rat sciatic nerve the synthesis and accumulation of soluble, extra-cellular, 37 kDa protein is increased. This protein, which accumulates in the extracellular space of the injured nerve, accounts for nearly 5% of the total soluble pool of protein in an injured nerve 3 weeks after injury. 8 weeks after injury, when regeneration is nearly complete, this accumulated pool returns to control levels, yet if regeneration is blocked synthesis of the 37 kDa protein remains high. Recently this 37 kDa protein has been shown to be nearly identical to apolipoprotein E, the protein component of various lipoprotein particles. This finding suggests a role for the 37 kDa protein in cholesterol and lipid transport and metabolism during nerve repair within the nervous system, functions that have been ascribed to apo E in serum. Results are presented here describing the purification of the nerve injury induced 37 kDa protein and the subsequent production of specific rabbit antisera directed against it. By centrifugation analysis in a sucrose gradient, a native mass of 37 kDa was determined, revealing the 37 kDa protein's monomeric, native structure. Additionally injections of [35S]methionine directly into the injured nerve allowed 1) a comparison of 37 kDa synthesis in vivo versus in vitro and 2) an examination of the presence or absence of retrogradely transported 37 kDa protein. The in vitro and in vivo collected material were found to share identical 2-dimensional electrophoretic mobilities, and no appreciable amount of transported 37 kDa protein was found in proximal regions of the injured nerve.

Lipoprotein uptake by neuronal growth cones in vitro.

Macrophages that rapidly enter injured peripheral nerve synthesize and secrete large quantities of apolipoprotein E. This protein may be involved in the redistribution of lipid, including cholesterol released during degeneration, to the regenerating axons. To test this postulate, apolipoprotein E-associated lipid particles released from segments of injured rat sciatic nerve and apolipoprotein E-containing lipoproteins from plasma were used to determine whether sprouting neurites, specifically their growth cones, possessed lipoprotein receptors. Pheochromocytoma (PC12) cells, which can be stimulated to produce neurites in vitro, were used as a model system. Apolipoprotein E-containing lipid particles and lipoproteins, which had been labeled with fluorescent dye, were internalized by the neurites and their growth cones; the unmetabolized dye appeared to be localized to the lysosomes. The rapid rate of accumulation in the growth cones precludes the possibility of orthograde transport of the fluorescent particles from the PC12 cell bodies. Thus, receptor-mediated lipoprotein uptake is performed by the apolipoprotein B,E(LDL) (low density lipoprotein) receptors, and in the regenerating peripheral nerve apolipoprotein E may deliver lipids to the neurites and their growth cones for membrane biosynthesis.

Expression of apolipoprotein E during nerve degeneration and regeneration.

A 37-kDa glycoprotein has been described recently, whose synthesis is dramatically increased after injury of the rat sciatic and optic nerves. Cells in the nerve sheath, distal to the site of injury, produce and secrete large amounts of this protein, so that by 3 weeks after injury, it represents 2-5% of the total soluble extracellular protein in the regenerating sciatic nerve sheath, although it fails to accumulate in damaged optic nerve. Results presented here reveal extensive homology between the 37-kDa nerve injury-induced protein and a well-studied serum protein, apolipoprotein E (apoE), that is involved in lipid and cholesterol metabolism and that has been shown recently to be present in adult and developing rat astroglia. Both proteins have identical isoelectric focusing points and similar molecular masses. Antibodies raised against the 37-kDa protein recognize apoE and anti-apoE serum crossreacts with the 37-kDa protein. Sequence data for two 14 amino acid stretches of the 37-kDa protein match identical regions of apoE. These data suggest that the 37-kDa protein is identical to serum apoE and that it could have similar functions to the latter. In the nervous system, for example, it may be involved in the mobilization and reutilization of lipid in the repair, growth, and maintenance of myelin and axonal membranes, both during development and after injury.

Expression of specific sheath cell proteins during peripheral nerve growth and regeneration in mammals.

Protein synthesis in the nerve sheath of injured as well as intact mature and developing sciatic nerves from rat and rabbit was investigated by incubating segments of nerve with [35S]methionine in vitro. The composition of labeled proteins under the different conditions of nerve growth was analyzed by two-dimensional gel electrophoresis and fluorography. The expression of six secreted proteins in rat sciatic nerve with the apparent molecular weights of 70,000 (70 kD), 54,000 (54 kD), 51,000 (51 kD), 39,000 (39 kD), 37,000 (37 kD), and 30,000 (30 kD) was of particular interest because of the correlation of their synthesis and secretion with aspects of nerve growth and regeneration. The synthesis of the 37-kD protein was significantly stimulated during both sciatic nerve development as well as regeneration but not in the intact mature nerve. The expression of this protein appears to be regulated by signal(s) from the axon but not the target. The 70-kD protein was exclusively synthesized in response to axotomy, thus confining its role to some aspect(s) of nerve repair. In contrast, the 54- and 51-kD proteins were expressed in the intact mature nerve sheath. Their synthesis and release was rapidly inhibited upon axotomy but returned to normal or higher levels towards the end of sciatic nerve regeneration, suggesting a role in the maintenance of the integrity of the mature (nongrowing) rat nerve. The 39- and 30-kD proteins were only transiently synthesized within the first week after axotomy. Two proteins with the apparent molecular masses of 70 and 37 kD were synthesized in denervated rabbit sciatic nerve. The similar molecular weights, net charges, and time-courses of induction suggest a homology between these proteins in rabbit and rat, indicating common molecular responses of peripheral nerve sheath cells to axon injury in both mammalian species.

Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells.

Triton X-100 solubilizes membranes of PC12 cells and leaves behind a nucleus and an array of cytoskeletal filaments. Nerve growth factor (NGF) receptors (10% of those found in intact cells) are associated with this Triton X-100-insoluble residue. Two classes of NGF receptors are found on PC12 cells which display rapid and slow dissociating kinetics. Although rapidly dissociating binding is predominant (greater than 75%) in intact cells, the majority of binding to the Triton X-100 cytoskeleton is slowly dissociating (greater than 75%). Rapidly dissociating NGF binding on intact cells can be converted to a slowly dissociating form by the plant lectin wheat germ agglutinin (WGA). This lectin also increases the number of receptors which associate with the Triton X-100 cytoskeleton by more than 10-fold. 125I-NGF bound to receptors can be visualized by light microscopy autoradiography in Triton X-100-insoluble residues of cell bodies, as well as growth cones and neurites. The WGA-induced association with the cytoskeleton, however, is not specific for the NGF receptor, since greater than 90% of cell surface glycoprotein receptors for WGA become associated with Triton X-100-insoluble material at lectin concentrations greater than 33 micrograms/ml. Concentrations of WGA which change the Triton X-100 solubility of membrane glycoproteins are similar to those required to alter the kinetic state of the NGF receptor. Both events may be related to the crossbridging of cell surface proteins induced by this multivalent lectin.

Nerve growth factor-treated, neurite-bearing PC12 cells continue to synthesize DNA.

Cultures of rat pheochromocytoma (PC12) cells treated with beta-nerve growth factor (NGF) for up to 15 days continue to synthesize DNA. The present study compares the extent of maintained DNA synthesis in cells with and without processes and asks whether the observed DNA synthesis in differentiated PC12 cells reflects either the continued division of the cells or the formation of polyploid cells, or both. PC12 cells were grown on tissue coverslips for various lengths of time with or without 50 ng/ml of beta-NGF and then assayed for DNA synthesis by [3H]thymidine labeling and autoradiography. In 8-day-old control cultures (no NGF), 30% of the cells had labeled nuclei after a 2-hr [3H]thymidine pulse. In contrast, in cultures treated for 8 days with NGF, only 7% of the cells were labeled (i.e., still synthesizing DNA). The fractions of process-bearing and non-process-bearing cells with labeled nuclei were identical. Even after 14 days in NGF, 7% of the cells with neurites were still synthesizing DNA during any 2-hr period. With continuous [3H]thymidine labeling in the presence of NGF from 8 to 13 days, nearly 70% of the cells with neurites were labeled. The presence of neurites induced by NGF does not preclude continued (albeit reduced) DNA synthesis in these PC12 cells. To determine the fate of this newly synthesized DNA, nuclei extracted from NGF-treated PC12 cells were analyzed for the cellular distribution of DNA by combined propidium iodine staining and flow microfluorimetry. NGF treatment resulted in a 3-fold increase in the number of G2+M/4N cells along with the appearance of 8N cells.(ABSTRACT TRUNCATED AT 250 WORDS)