Enhanced visualization of axonopathy in EAE using thy1-YFP transgenic mice.

It is widely accepted that chronic disabilities in multiple sclerosis (MS) patients are due in part to neuronal damage. The central aim of this study was to characterize axonal disruption in the spinal cord of mice with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE), a model of progressive MS. To accomplish this goal, we induced MOG-EAE in thy1-yellow fluorescent (thy-YFP)-transgenic mice in which all spinal motorneurons express the YFP reporter protein. We demonstrate that a build-up of YFP fluorescence occurs in profiles reminiscent of tortuous fragmented axons and axonal spheroids/globules as seen in various neurodegenerative/neuroinflammatory diseases. Approximately two-thirds of these damaged axons were decorated by the monoclonal antibody SMI 32, which recognizes hypophosphorylated neurofilament-H (hypoP-NF-H), an established marker of CNS axonal pathology. Unexpectedly, one third of damaged axons were hypoP-NF-H negative but could be visualized by their expression of the YFP transgene, whilst the remaining profiles were hypoP-NF-H positive but did not exhibit YFP fluorescence. Thus, using YFP transgenic mice in conjunction with hypoP-NF-H immunoreactivity provides a more comprehensive depiction of axonopathy in the ventral-lateral aspect of lumbosacral spinal cord in MOG-EAE. When YFP fluorescence was used in conjunction with a monoclonal antibody that recognizes CD11b; a marker of subsets of inflammatory cells, we were able to discern evidence of an early inflammatory attack on white matter axons. Finally, we show the accumulation of hyperphosphorylated neurofilament-H (hyperP-NF-H) expression in YFP+, lesioned WM areas and in a subpopulation of neuronal perikarya in the lumbar spinal cords of EAE mice.

Motor neuron pathology in experimental autoimmune encephalomyelitis: studies in THY1-YFP transgenic mice.

Using adult male C57BL/6 mice that express a yellow fluorescent protein transgene in their motor neurons, we induced experimental autoimmune encephalomyelitis (EAE) by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG peptide) in complete Freund's adjuvant (CFA). Control mice of the same transgenic strain received CFA without MOG peptide. Early in the course of their illness, the EAE mice showed lumbosacral spinal cord inflammation, demyelination and axonal fragmentation. By 14 weeks post-MOG peptide, these abnormalities were much less prominent, but the mice remained weak and, as in patients with progressive multiple sclerosis, spinal cord atrophy had developed. There was no significant loss of lumbar spinal cord motor neurons in the MOG peptide-EAE mice. However, early in the course of the illness, motor neuron dendrites were disrupted and motor neuron expression of hypophosphorylated neurofilament-H (hypoP-NF-H) immunoreactivity was diminished. By 14 weeks post-MOG peptide, hypoP-NF-H expression had returned to normal, but motor neuron dendritic abnormalities persisted and motor neuron perikaryal atrophy had appeared. We hypothesize that these motor neuron abnormalities contribute to weakness in this form of EAE and speculate that similar motor neuron abnormalities are present in patients with progressive multiple sclerosis.

Glial growth factor-2 promotes the survival, migration and bromodeoxyuridine incorporation of mammalian neural crest cells in caudal neural tube explant cultures.

Using an in vitro assay system, we found that GGF-2 increases the number of nascent trunk neural crest cells (NCC) present in the dorsal outgrowth derived from E12 caudal neural tube explants. Data is presented which suggests that this increased outgrowth was due to a combination of GGF-2 mediated effects, including its ability to promote (A) NCC survival by decreasing the percentage of NCC that undergo cell death via a mechanism involving DNA fragmentation, (B) the initial phases of NCC migration, (C) mitosis of peripherally migrating NCC. We also show that GGF-2 can promote the long-term survival of NCC in the absence of the neural tube. An immunohistochemical analysis indicates that NCC express erbB-2 and erbB-4 neuregulin receptors.

Defensin promotes the binding of lipoprotein(a) to vascular matrix.

Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that alpha-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that alpha-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.

The spatial and temporal expression of HNK-1 by myogenic and skeletogenic cells in the embryonic rat.

The existence of phenotypic differences within a population of cells provides evidence for discrete stages in cellular differentiation and/or identifies subsets of cells with unique functional properties. The monoclonal antibody HNK-1 has been widely shown to identify subpopulations of cells in the developing nervous system. In this paper we focus on the developmental expression of HNK-1 immunoreactivity by derivatives of somitic (paraxial) mesoderm. We show that between embryonic day 12 and 14 (E12-E14) the HNK-1 epitope is transiently expressed by postmitotic myotomal cells. In E14-E17 developing vertebral columns (which are derived from somitic sclerotomal cells), HNK-1 immunolabeling was expressed by subpopulations of skeletogenic cells, including perinotochordal cells associated with the forming annulus fibrosus and cells within or adjacent to the perichondrium. Chondrocytes within forming centra and vertebral arches did not exhibit HNK-1 immunoreactivity. These results, taken together, show that the expression of the HNK-1 epitope can be used to identify subsets of myogenic and skeletogenic cells both spatially and temporally in the developing rat.

Multiple signaling pathways regulate cell surface expression and activity of the excitatory amino acid carrier 1 subtype of Glu transporter in C6 glioma.

Neuronal and glial sodium-dependent transporters are crucial for the control of extracellular glutamate levels in the CNS. The regulation of these transporters is relatively unexplored, but the activity of other transporters is regulated by protein kinase C (PKC)- and phosphatidylinositol 3-kinase (PI3K)-mediated trafficking to and from the cell surface. In the present study the C6 glioma cell line was used as a model system that endogenously expresses the excitatory amino acid carrier 1 (EAAC1) subtype of neuronal glutamate transporter. As previously observed, phorbol 12-myristate 13-acetate (PMA) caused an 80% increase in transporter activity within minutes that cannot be attributed to the synthesis of new transporters. This increase in activity correlated with an increase in cell surface expression of EAAC1 as measured by using a membrane-impermeant biotinylation reagent. Both effects of PMA were blocked by the PKC inhibitor bisindolylmaleimide II (Bis II). The putative PI3K inhibitor, wortmannin, decreased L-[3H]-glutamate uptake activity by >50% within minutes. Wortmannin decreased the Vmax of L-[3H]-glutamate and D-[3H]-aspartate transport, but it did not affect Na+-dependent [3H]-glycine transport. Wortmannin also decreased cell surface expression of EAAC1. Although wortmannin did not block the effects of PMA on activity, it prevented the PMA-induced increase in cell surface expression. This trafficking of EAAC1 also was examined with immunofluorescent confocal microscopy, which supported the biotinylation studies and also revealed a clustering of EAAC1 at cell surface after treatment with PMA. These studies suggest that the trafficking of the neuronal glutamate transporter EAAC1 is regulated by two independent signaling pathways and also may suggest a novel endogenous protective mechanism to limit glutamate-induced excitotoxicity.

Effects of FGF-1 and FGF-2 on GD3 immunoreactive spinal neuroepithelial cells.

Embryonic central nervous system neuroepithelial cells are a transient population of cells that give rise to neuronal and glial progenitors. In the E12-E16 embryonic rat spinal neural tube we have identified neuroepithelial cells as radially oriented cells expressing the GD3 ganglioside as recognized by the monoclonal anti-GD3 ganglioside antibodies, R24 and LB1. In vitro, neuroepithelial cells, which migrate from the ventral aspect of E12 rat lumbosacral neural tube explants, also express GD3 ganglioside immunoreactivity, thus permitting their distinction from neural crest cells (NCC) which migrate from the dorsal aspect of such explants. Fibroblast growth factor-1 (FGF-1, acidic FGF) and FGF-2 (basic FGF) increase the migration of neuroepithelial cells and the extent to which they incorporate the thymidine analogue bromodeoxyuridine (BrdU). They do not, however, alter the rate at which these migrating neuroepithelial cells undergo cell death. Previous observations established the actions of FGF-1 and FGF-2 on neuronal and glial cells. The present study indicates that these growth factors also influence the motility and proliferation of progenitor cells at a developmental stage which precedes their divergence into neuronal and glial lineages.

Periaxin expression in myelinating Schwann cells: modulation by axon-glial interactions and polarized localization during development.

Periaxin is a newly described protein that is expressed exclusively by myelinating Schwann cells. In developing nerves, periaxin is first detected as Schwann cells ensheathe axons, prior to the appearance of the proteins that characterize the myelin sheath. Periaxin is initially concentrated in the adaxonal membrane (apposing the axon) but, during development, as myelin sheaths mature, periaxin becomes predominately localized at the abaxonal Schwann cell membrane (apposing the basal lamina). In permanently axotomized adult nerves, periaxin is lost from the abaxonal and adaxonal membranes, becomes associated with degenerating myelin sheaths and is phagocytosed by macrophages. In crushed nerves, in which axons regenerate and are remyelinated, periaxin is first detected in the adoxonal membrane as Schwann cells ensheathe regenerating axons, but again prior to the appearance of other myelin proteins. Periaxin mRNA and protein levels change in parallel with those of other myelin-related genes after permanent axotomy and crush. These data demonstrate that periaxin is expressed by myelinating Schwann cells in a dynamic, developmentally regulated manner. The shift in localization of periaxin in the Schwann cell after completion of the spiralization phase of myelination suggests that periaxin participates in membrane-protein interactions that are required to stabilize the mature myelin sheath.

Acidic and basic fibroblast growth factors delay the maturation of neural crest-derived neurons.

Neural crest (NC) cultures were prepared from lumbosacral segments of 12 day rat embryos and maintained in a defined medium. Post-mitotic, flat, neurofilament+ neurons with broad neuritic processes ('nascent neurons') appeared within 24 h. Timing of the next stage in neuronal differentiation, the formation of bipolar, phase-bright cells that bound tetanus toxin with long, slender neurites ('bipolar neurons'), was markedly influenced by acidic or basic fibroblast growth factor (FGF). The transition from nascent to bipolar neuron occurred several days prematurely in medium without added FGF, but took place with a time-course like that in vivo when 10 ng/ml of acidic or basic FGF was added.

Protein growth factor requirements of rat neural crest cells.

Nascent neural crest cells derived from explanted E12 embryonic rat caudal neural tubes were used as an assay system to investigate the effects of fibroblast growth factors on neural crest cell (NCC) survival, proliferation, migration, and differentiation. In vitro and in vivo all NCC express low affinity nerve growth factor receptors (p75-LNGFR), whereas a subpopulation of NCC expresses the carbohydrate epitope recognized by the monoclonal antibody HNK-1 (Bannerman and Pleasure, manuscript in preparation). Both acidic and basic fibroblast growth factor (FGF) promoted the survival of proportionally greater numbers of p75-LNGF+/HNK-1- than P75-LNGFR+/HNK-1+ NCC. An as yet uncharacterized factor present in neural tube-conditioned medium was also required for NCC survival. Mitosis was frequent in those NCC closest to the neural tube, less so as the cells migrated away. Neither basic nor acidic fibroblast growth factor (FGF) influenced rates of NCC mitosis in either of these locations, nor did these FGFs alter the rate at which nascent NCC migrated away from the neural tube. However, acidic and basic FGFs did delay the differentiation of neural crest derived neurons in the cultures. FGF is abundant in the embryonic rat neural crest outgrowth zone, and the present study strongly supports an essential role for FGF in early development of the mammalian neural crest.

Electrophysiologic and molecular properties of cultured enteric glia.

Enteric glia, the support cells of myenteric ganglia, have been widely studied with respect to their morphology and immunohistochemical phenotype, but little is known about their functional properties. We developed a method for the amplification of enteric glia from newborn guinea pigs to further characterize these cells. Treatment with a combination of basic fibroblast growth factor and the adenylate cyclase activator, cholera toxin, permitted expansion of enteric glial cultures to confluence and serial passage for up to 8 months. The long-term cultured cells retained expression of 1) S100 protein, 2) GD3 ganglioside recognized by the monoclonal antibody LB1, and 3) the gene encoding glutamine synthetase. The electrophysiologic properties of cultured enteric glia were studied under whole-cell patch clamp conditions. Most cells expressed "delayed rectifier"-type potassium currents, and some also demonstrated tetrodotoxin-sensitive sodium currents. Other subsets of voltage-dependent potassium currents, calcium currents, and glutamate-gated currents were not demonstrable.

Control of peripheral glial cell proliferation: enteric neurons exert an inhibitory influence on Schwann cell and enteric glial cell DNA synthesis in culture.

Neuronal membranes from rat dorsal root ganglia provide a mitogenic signal to cultured Schwann cells and it has been suggested this is an important factor in regulating Schwann cell numbers during development. In this study, the influence of enteric neurons on the DNA synthesis of both Schwann cells and enteric glia has been investigated as well as the effect of axonal membrane fractions (axolemma) on enteric glia. The proliferation rate of rat Schwann cells and enteric glia was assessed in culture using [3H]thymidine uptake and autoradiography in combination with immunolabelling to identify cell types. When purified rat Schwann cells were co-cultured with guinea pig enteric neurons, their DNA synthesis rate was reduced compared with control cultures of pure Schwann cells or Schwann cells not close to neurites or neuronal cell bodies. Nevertheless, in accordance with previous findings that sensory neurons stimulate Schwann cell division, these Schwann cells increased their DNA synthesis rate when in contact with neurites from purified guinea pig or adult rat dorsal root ganglion neurons and on exposure to bovine axolemmal fractions. The enteric neurons also suppressed the DNA synthesis of enteric glia in co-cultures of purified enteric neurons and enteric glia, while bovine axolemma stimulated their DNA synthesis. These results indicate that a mitotic inhibitory signal is associated with enteric neurons and can exert its effect on both Schwann cells and enteric glia, and that enteric glia, like Schwann cells, are stimulated to divide by axolemmal fractions. It thus seems possible that during development glial cell numbers in the peripheral nervous system may be controlled by both positive and negative regulators of cell growth.

Antigenic markers and laminin expression in cultured enteric neural cells.

In this study, polyclonal and monoclonal antibodies have been used in conjunction with standard immunohistochemical methods to define markers which can be used to identify and study the main cell types present in the outgrowth area of explant cultures of myenteric plexus from newborn guinea pig. We show that all of the neurons binds antibodies to the glycoprotein Thy-1 and the antibody A2B5 which recognizes gangliosides. All enteric glial cells bind antibodies to the calcium-binding protein S100, and the A2B5 monoclonal antibody and ca. 95% of glia are labeled by the antibody LB1, which recognizes the GD3 ganglioside. Most fibroblasts are labelled by antibodies to Thy-1 and the matrix glycoprotein fibronectin. Thus enteric neurons can be defined serologically as Thy-1+/A2B5+/S100- cells; enteric glia as S100+ cells and fibroblasts as A2B5-/S100-cells. The markers have been used to demonstrate that laminin is made by both enteric glia and fibroblasts. They have also been used to show that ca. 5% of neurons and less than 5% of enteric glia bind the antibody Leu 7 (HNK-1, L2), thus revealing the subpopulations of neurons and glia show differential cell surface expression of the carbohydrate epitope recognized by the antibody. In the accompanying paper, we demonstrate that two of the antibodies (LB1 and Thy-1) can be used to generate purified populations of neurons and glia.

Establishment and properties of separate cultures of enteric neurons and enteric glia.

In this paper methods are described for the preparation of two types of culture derived from myenteric explants: (a) highly enriched neuronal cell cultures, and (b) purified glial cells (greater than 98%). Both procedures combine the technique of antibody complement-mediated cytolysis with the use of an antimitotic agent. Immunohistochemical methods were used to compare the purified cells to their counterparts in mixed cultures (see accompanying paper). Antibodies to the glycoprotein Thy-1 and the monoclonal antibody A2B5 which recognizes gangliosides, labelled the cell surface of all enteric neurons in enriched cultures while subpopulations of the neurons expressed the Leu 7 carbohydrate epitope, the neurotransmitter 5-hydroxytryptamine and the neuropeptides substance P, methionine-enkephalin and vasoactive intestinal polypeptide. Autoradiographic experiments show that a subpopulation of enriched neurons exhibit high-affinity uptake sites for gamma-[3H]aminobutyric acid (GABA). All purified enteric glia continue to express the calcium binding protein S100, the basement membrane glycoprotein laminin and the antigens recognized by the A2B5 antibody, and subpopulations of glia are labelled by the monoclonal antibodies LB1 which binds to GD3 gangliosides, and Leu 7. Thus enteric neurons and glia can survive independently of each other and express molecular properties which are present in cultures normally containing both cell types.

Analysis of enteric neurons, glia and their interactions using explant cultures of the myenteric plexus.

The enteric nervous system (ENS) of the gastrointestinal tract is the largest and most complicated division of the peripheral nervous system. The ENS possesses reflex pathways composed of motor neurons, interneurons and sensory neurons which act in an integrated fashion together with input from the central nervous system to control gut function. The neurons, morphologically and electrophysiologically a very heterogeneous group containing a large number of different proven and putative neurotransmitters, are intimately associated with enteric glia, which both at the morphological and molecular level resemble astrocytes. In this review we describe how explant cultures from the ENS have been used to investigate the neurochemical, molecular and electrophysiological characteristics of ENS neurons, the molecular properties of enteric glia and their interactions with one another.

Light microscopic immunolocalization of laminin, type IV collagen, nidogen, heparan sulphate proteoglycan and fibronectin in the enteric nervous system of rat and guinea pig.

The localization of the extracellular matrix components laminin, fibronectin and type IV collagen in the enteric nervous system and the surrounding smooth muscle was investigated by immunohistochemical methods, using tissue sections of rat and guinea pig large intestine. None of these molecules were detectable inside the enteric ganglia. In contrast, they were easily demonstrable in association with the basement membrane of satellite cells within sensory and sympathetic ganglia. All of these molecules were, however, present in or nearby the basement membrane that surrounds each enteric ganglion. This agrees with previous ultrastructural observations that, in small mammals, neither basement membranes nor large connective tissue spaces are found inside enteric ganglia. The matrix molecules under study were also detected in the basement membrane of the nearby smooth muscle cells that make up the muscle layer of the gut wall. Fibronectin was frequently observed as a broad staining pattern suggesting its localization in the lamina reticularis rather than in the lamina densa. In addition, nidogen and heparan sulphate proteoglycan were demonstrated in the basement membrane of both enteric ganglia and Schwann cells.