Establishment and neurite outgrowth properties of neonatal and adult rat olfactory bulb glial cell lines

Two glial cell types surround olfactory axons and glomeruli in the olfactory bulb (OB) and may influence synapse development and regeneration. OB astrocytes resemble type-1 astrocytes, and OB ensheathing cells resemble non-myelinating Schwann cells. We have produced clonal OB astrocyte and ensheathing cell lines from rat neonatal and adult OB cultures by SV40 large T antigen transduction. These cell lines have been characterized by morphology, growth characteristics, immunophenotype, and ability to promote neurite outgrowth in vitro. Neonatal and adult ensheathing cell lines were found to support higher neurite outgrowth than OB astrocyte lines. Neonatal OB astrocyte lines were of two types, high and low outgrowth support. The low support astrocyte lines express J1 and a chondroitin sulfate-containing proteoglycan as do astrocytes encircling the neonatal glomeruli in vivo. The adult OB astrocyte cell lines supported lower levels of outgrowth than adult ensheathing cell lines. These results are consistent with a positive role for ensheathing cells in OB synapse regeneration, in vivo. Further, based on our results, we hypothesize that ensheathing cells and high-outgrowth astrocytes facilitate axon growth in vivo, while low outgrowth astrocytes inhibit axon growth and may facilitate glomerulus formation.     

MAG and MOG enhance neurite outgrowth of embryonic mouse spinal cord neurons

Myelin-associated glycoprotein (MAG) inhibits neurite outgrowth of postnatal spinal cord neurons, but its effect on embryonic neurons is unknown. The effect on neurite outgrowth of another myelin protein, myelin-oligodendrocyte glycoprotein (MOG) is also unknown. We determined the effect of MAG and MOG on embryonic day 17 spinal cord neurons, which were cultured on MAG, MOG or control transfected CHO cells. Neurite outgrowth was examined and both total neurite length and longest neurite length were significantly enhanced by both MAG and MOG. These findings show that, in contrast to postnatal spinal cord neurons, MAG can enhance neurite outgrowth of embryonic spinal cord neurons. In addition, another myelin protein, MOG, can also modulate neurite outgrowth.     

Thrombin induced inhibition of neurite outgrowth from dorsal root ganglion neurons

Adrenalectomy (ADX) is known to block the acquisition of intravenous cocaine self-administration. A previous study therefore examined whether ADX decreases sensitivity of the 'brain reward system' in general, or its response to cocaine in particular, by measuring thresholds for intracranial self-stimulation with and without concurrent cocaine administration. ADX had no effect on thresholds for lateral hypothalamic self-stimulation (LHSS) and did not alter the cocaine dose-response curve for lowering the LHSS threshold. This result suggested that ADX does not affect sensitivity of the brain reward system. However, medial prefrontal cortex (MPFC) appears to be an important site in the mediation of cocaine reinforcing effects, and MPFC self-stimulation (MPFCSS) is mediated by a neural substrate that is largely independent of that which mediates LHSS. The present study therefore assessed whether ADX diminishes cocaine facilitation of MPFCSS. It was found that the threshold-lowering effect of cocaine (5.0, 10.0 and 20.0 mg/kg, i.p. ) did not differ between ADX rats maintained on 0.7% saline, ADX rats maintained on corticosterone (50 microg/ml) in 0.7% saline, and sham-operated controls. However, there was a trend toward desensitization of MPFCSS, itself, following ADX in the group that did not receive corticosterone supplementation. Based on this observation, and the similar responses of MPFCSS and cocaine self-administration to noncontingent priming stimulation, stress, and NMDA receptor antagonism, it is speculated that acquisition of MPFCSS and cocaine self-administration may be dependent upon a common sensitization process that is regulated by corticosterone.     

Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation

Increasing evidence indicates several roles for thrombin-like serine proteases and their cognate inhibitors (serpins) in normal development and/or pathology of the nervous system. In addition to its prominent role in thrombosis and/or hemostasis, thrombin inhibits neurite outgrowth in neuroblastoma and primary neuronal cells in vitro, prevents stellation of glial cells, and induces cell death in glial and neuronal cell cultures. Thrombin is known to act via a cell surface protease-activated receptor (PAR-1), and recent evidence suggests that rodent neurons express PAR-1. Previously, we have shown that the thrombin inhibitor, protease nexin-1, significantly prevents neuronal cell death both in vitro and in vivo. Here we have examined the effects of human alpha-thrombin and the presence and/or activation of PAR-1 on the survival and differentiation of highly enriched cultures of embryonic chick spinal motoneurons. We show that thrombin significantly decreased the mean neurite length, prevented neurite branching, and induced motoneuron death by an apoptosis-like mechanism in a dose-dependent manner. These effects were prevented by cotreatment with hirudin, a specific thrombin inhibitor. Treatment of the cultures with a synthetic thrombin receptor-activating peptide (SFLLRNP) mimicked the deleterious effects of thrombin on motoneurons. Furthermore, cotreatment of the cultures with inhibitors of caspase activities completely prevented the death of motoneurons induced by either thrombin or SFLLRNP. These findings indicate that (1) embryonic avian spinal motoneurons express functional PAR-1 and (2) activation of this receptor induces neuronal cell degeneration and death via stimulation of caspases. Together with previous reports, our results suggest that thrombin, its receptor(s), and endogenous thrombin inhibitors may be important regulators of neuronal cell fate during development, after injury, and in pathology of the nervous system.     

Mouse cortical neurones lacking APP show normal neurite outgrowth and survival responses in vitro

We have cultured neurones from the developing cortex of mice that have had the amyloid precursor protein gene deleted (APP-null). Neurones cultured for a period of 24 h show similar neurite outgrowth and survival responses to wild-type neurones. Similar neurite outgrowth responses were also seen when neurones from APP-null mice were treated with a neurotrophic peptide derived from the APP sequence and compared with wild-type neurones. Finally, cortical cultures derived from APP-null mice showed similar survival responses to the toxic amyloid-beta peptide.     

Neurturin and GDNF promote proliferation and survival of enteric neuron and glial progenitors in vitro

Signaling through the c-Ret tyrosine kinase and the endothelin B receptor pathways is known to be critical for development of the enteric nervous system. To clarify the role of these receptors in enteric nervous system development, the effect of ligands for these receptors was examined on rat enteric neuron precursors in fully defined medium in primary culture. In this culture system, dividing Ret-positive cells differentiate, cluster into ganglia containing neurons and enteric glia, and create extensive networks reminiscent of the enteric plexus established in vivo. Glial cell-line-derived neurotrophic factor (GDNF) and neurturin both potently support survival and proliferation of enteric neuron precursors in this system. Addition of either neurturin or GDNF to these cultures increased the number of both neurons and enteric glia. Persephin, a third GDNF family member, shares many properties with neurturin and GDNF in the central nervous system and in kidney development. By contrast, persephin does not promote enteric neuron precursor proliferation or survival in these cultures. Endothelin-3 also does not increase the number of enteric neurons or glia in these cultures.     

Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.     

The brain chondroitin sulfate proteoglycan brevican associates with astrocytes ensheathing cerebellar glomeruli and inhibits neurite outgrowth from granule neurons

Brevican is a nervous system-specific chondroitin sulfate proteoglycan that belongs to the aggrecan family and is one of the most abundant chondroitin sulfate proteoglycans in adult brain. To gain insights into the role of brevican in brain development, we investigated its spatiotemporal expression, cell surface binding, and effects on neurite outgrowth, using rat cerebellar cortex as a model system. Immunoreactivity of brevican occurs predominantly in the protoplasmic islet in the internal granular layer after the third postnatal week. Immunoelectron microscopy revealed that brevican is localized in close association with the surface of astrocytes that form neuroglial sheaths of cerebellar glomeruli where incoming mossy fibers interact with dendrites and axons from resident neurons. In situ hybridization showed that brevican is synthesized by these astrocytes themselves. In primary cultures of cerebellar astrocytes, brevican is detected on the surface of these cells. Binding assays with exogenously added brevican revealed that primary astrocytes and several immortalized neural cell lines have cell surface binding sites for brevican core protein. These cell surface brevican binding sites recognize the C-terminal portion of the core protein and are independent of cell surface hyaluronan. These results indicate that brevican is synthesized by astrocytes and retained on their surface by an interaction involving its core protein. Purified brevican inhibits neurite outgrowth from cerebellar granule neurons in vitro, an activity that requires chondroitin sulfate chains. We suggest that brevican presented on the surface of neuroglial sheaths may be controlling the infiltration of axons and dendrites into maturing glomeruli.     

Laminin-like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro

OBJECTIVE: To review data supporting the hypothesis that syndrome X plays a major role in the pathogenesis of coronary artery disease (CAD), and the effects of lifestyle factors and pharmacologic interventions on insulin, other metabolic parameters, and outcomes. DATA SOURCES: MEDLINE (January 1966-August 1997) and Current Contents database searches identified applicable English-language experimental trials, epidemiologic studies, reviews, and editorials. STUDY SELECTION AND DATA EXTRACTION: Studies that were included addressed the role of insulin resistance and hyperinsulinemia in the pathogenesis of CAD or the effects of lifestyle factors and pharmacologic interventions on metabolic parameters and outcomes. DATA SYNTHESIS: The main characteristics of syndrome X are hyperinsulinemia and insulin resistance. These result in secondary syndrome X features, including hyperglycemia, increased very-low-density lipoprotein concentrations, decreased high-density lipoprotein cholesterol, and hypertension. Insulin resistance is worsened by obesity, and insulin has been shown to contribute to the development of hypertension. Other studies demonstrate that smoking adversely affects glucose and insulin concentrations. Animal studies have linked hyperinsulinemia and atherogenesis. These animal data have been confirmed by several large prospective and population studies that have identified associations between hyperinsulinemia and CAD. CONCLUSIONS: Strong evidence links insulin resistance and hyperinsulinemia to CAD. Lifestyle modifications play an important role in decreasing cardiovascular risk, and clinicians should strongly encourage such changes. Clinicians must also carefully consider the effects of antihypertensive, antihyperglycemic, and antidyslipidemic agents on patients' metabolic profiles when choosing appropriate therapeutic regimens. However, outcome data on many potentially beneficial agents, including calcium antagonists, alpha 1-adrenergic antagonists, angiotensin-converting enzyme inhibitors, metformin, acarbose, and troglitazone, are not yet available.     

Electrically induced changes in Ca2+ in Helisoma neurons: regional and neuron-specific differences and implications for neurite outgrowth

The present experiments addressed the questions of how electrical stimulation influenced the magnitude, time course, and regional levels of free intracellular calcium of different identified neurons. The calcium concentration in the growth cones, neurites and cell bodies of Helisoma buccal neurons B4 and B19 was measured while somata were electrically stimulated via an intracellular electrode. The findings showed that calcium levels in B4 and B19 increased monotonically with increasing stimulation frequency. However, the range of calcium levels evoked by electrical stimulation differed significantly for each type of neuron. The greater increase in calcium concentration in B4 was correlated with its longer duration action potential compared to B19. The increase in calcium concentration was much smaller in the cell bodies than in the growth cones and neurites. Extending the duration of the B19 action potential produced a sixfold increase in the change in calcium concentration at 2 Hz stimulation. Under conditions where the electrical stimulation produced a calcium concentration of < 160 nM, the elevated level of free intracellular calcium remained constant. When calcium concentration increased above 200 nM in both identified neurons, an initial peak concentration was followed by a decline to a lower concentration suggesting increased calcium buffering occurring above 200 nM. By correlating the calcium concentration data herein with growth data from a previous study, we suggest that specific calcium levels that influence neurite outgrowth may differ widely between neurons.     

Limbic system-associated membrane protein (LAMP) induces neurite outgrowth and intracellular Ca2+ increase in primary fetal neurons

The ability of cell adhesion molecules (CAMs) to transduce cell surface signals into intracellular responses is critical for developing neurons, particularly during axonal pathfinding and targeting. It has been suggested that different CAMs can promote neuronal outgrowth via activation of common neuronal CAM-specific second-messenger pathways, although the elements involved in this cascade could differ. Limbic system-associated membrane protein (LAMP), a member of the Ig superfamily, is a molecule that promotes cell adhesion and neurite outgrowth from specific populations of fetal neurons. In the present study, we show that LAMP can induce several types of calcium (Ca2+) signals. Neurite outgrowth is promoted if fetal hippocampal neurons are grown on lamp-transfected CHO cells. This LAMP-induced outgrowth of neurons is mediated in part through activation of L-type Ca channels. Application of soluble LAMP to cultures of fetal hippocampal neurons caused a sustained (up to 60 min) elevation of intracellular Ca2+ as measured by fluo-3 fluorescence on a confocal microscope. The number of responding hippocampal neurons was initially low, but increased with age in culture and the [Ca2+]i elevation was only partially decreased by an L-type Ca(2+)-channel blocker. In contrast, at all times in culture, only a small fraction of neurons from visual cortex responded to LAMP application and only with transient elevation of cytosolic Ca2+ (< 15 min). Based on these observations, LAMP appears to function primarily through homophilic interactions and acts in part by modulating intracellular Ca2+ levels during neurite outgrowth by increasing the Ca2+ influx through L-type calcium channels, but has additional effects on intracellular Ca2+ signaling at later developmental stages.     

Tenascin-C inhibits beta1 integrin-dependent cell adhesion and neurite outgrowth on fibronectin by a disialoganglioside-mediated signaling mechanism

We have previously shown that the extracellular matrix molecule tenascin-C inhibits fibronectin-mediated cell adhesion and neurite outgrowth by an interaction with a cellular RGD-independent receptor which interferes with the adhesion and neurite outgrowth promoting activities of the fibronectin receptor(s). Here we demonstrate that the inhibitory effect of tenascin-C on beta1integrin-dependent cell adhesion and neurite outgrowth is mediated by the interaction of the protein with membrane-associated disialogangliosides, which interferes with protein kinase C-related signaling pathways. First, in substratum mixtures with fibronectin, an RGD sequence-containing fragment of the molecule or synthetic peptide, tenascin-C inhibited cell adhesion and spreading by a disialoganglioside-dependent, sialidase-sensitive mechanism leading to an inhibition of protein kinase C. Second, the interaction of intact or trypsinized, i.e., cell surface glycoprotein-free, cells with immobilized tenascin-C was strongly inhibited by gangliosides or antibodies to gangliosides and tenascin-C. Third, preincubation of immobilized tenascin-C with soluble disialogangliosides resulted in a delayed cell detachment as a function of time. Similar to tenascin-C, immobilized antibody to GD2 (3F8) or sphingosine, a protein kinase C inhibitor, strongly inhibited RGD-dependent cell spreading. Finally, the degree of tenascin-C-induced inhibition of cell adhesion was proportional to the degree of disialoganglioside levels of expression by different cells suggesting the relevance of such mechanism in modulating integrin-mediated cell-matrix interactions during pattern formation or tumor progression.     

Molecular cloning and functional expression of gicerin, a novel cell adhesion molecule that binds to neurite outgrowth factor

Gicerin is an integral membrane glycoprotein of about 82 kd that is transiently expressed in the developing CNS. Gicerin was first identified as a binding protein for neurite outgrowth factor (NOF), a member of the laminin family of extracellular matrix proteins. By isolating and sequencing a gicerin cDNA, we have found that this protein is a novel member of the immunoglobulin superfamily. The deduced protein (584 amino acids) consists of five immunoglobulin-like loop structures in an extracellular domain, a single transmembrane region, and a short cytoplasmic tail. Cells transfected stably with gicerin cDNA adhered to NOF and aggregated with each other, indicating that gicerin exhibits both heterophilic and homophilic adhesion activities.     

Enhancement of neurite outgrowth by the soluble form of human L1 (neural cell adhesion molecule)

L1, a neural cell adhesion molecule, is involved in neurite outgrowth, migration and fasciculation. Although L1 is a membrane glycoprotein expressed on neural cells, the soluble form of L1 is generated in vivo by proteolysis. In the present study, a stable transfectant of Chinese hamster ovary (CHO) cells secreting human L1 without cytoplasmic and membrane spanning domains was generated, and the function of the secreted L1 was examined. Explants from embryonic chick brain stem were cultured on a substrate coated with polyethylenimine (PEI) alone, on substrate-bound L1 or in medium containing soluble L1. The neurites induced by L1, both the substrate-bound form and the soluble form, were 2-3 times longer than those cultured on PEI. The ability of the soluble L1 to induce neurite formation was slightly greater than that of the substrate L1. The present results demonstrated that neurite outgrowth was induced not only by substrate-bound L1 but also by soluble L1. Soluble L1 could be a pharmaceutical candidate for the promotion of nerve regeneration.     

The PI capsid region of Theiler's virus controls replication in mouse glial cell cultures

The GDVII strain of Theiler's virus is virulent. The DA strain is avirulent and can persist and initiate lesions of inflammatory demyelination in the CNS of susceptible strains of mice. Other, resistant strains of mice clear the infection. Replication of the GDVII and DA strains of Theiler's virus and their genetic recombinants R2, R3 and R4 were compared in mixed glial cell cultures derived from the mouse CNS. Differences were observed in the early rate of viral production. These mapped to the P1 capsid region of the viral genome. Viruses with GDVII P1 sequences produced virus and spread more rapidly than viruses with DA P1 sequences. GDVII virus infected greater numbers of cells than DA virus. Both strains of virus rapidly replicated at least to the level of translation in astrocytes (GFAP+), macrophage/microglial cells (F4/80+), oligodendrocytes (O4+) and bipotential precursor (A2B5+) cells. Early in infection many A2B5+ and GFAP+ cells were infected and destroyed. In contrast, O4+ cells were relatively resistant to cell-death. The cultures survived and produced virus over 14 days of study, at which time all 4 cell-type were present in the culture but < 1% of all the cells, the majority of which were O4+, expressed viral protein. Most of these infected O4+ cells retained a healthy morphology with extensive sheets of cytoplasm, suggesting that Theiler's virus infection of mature oligodendrocytes was non-destructive.