Yokichi Hayashi

Characterization of Chick Gizzard Extract That Promotes Neurite Outgrowth in Cultured Ciliary Neurons

Abstract: Chicken gizzard extract contains a macromolecule(s) that promotes the neurite outgrowth of dissociated neurons from the ciliary ganglia (CG) of chick embryos. The factor in gizzard extract was partially purified and estimated to be about 12S (M.W. 200,000‐300,000) on sucrose density gradient centrifugation. The neurite outgrowth of CG neurons by the factor strictly depends on the embryonal age. The maximal neurite outgrowth was observed when CG neurons were dissociated from the embryos younger than 10 days. After that time the response of CG neurons to the factor rapidly declined and was almost lost at day 14. The amount of factor in the gizzard began to increase rapidly from 12‐day‐old embryo and reached the maximal level at day 16, and thereafter a fairly steady level was maintained. When CG neurons were co‐cultured with rat myotubes, the ratio of muscle cells with synaptic responses (miniature end‐plate potentials) was significantly higher in the presence of gizzard factor than its absence. The results suggest that this factor acts as an external signal on CG neurons to form synaptic connections in vivo.

Etoposide induces programmed death in neurons cultured from the fetal rat central nervous system

The effects of etoposide on the death of neurons cultured from the central nervous system (CNS) of fetal rats were examined. The cultured neurons died in the presence of 1-40 micrograms/ml of etoposide, which is known to induce programmed death in some kinds of cells, and this cytotoxic effect was prevented by inhibition of protein synthesis and/or RNA synthesis. Furthermore, DNA degradation, including a ladder-like pattern, became evident in these neurons 3 h after incubation with etoposide (10 micrograms/ml), whereas cell death commenced after about 6 h. These results indicate that etoposide-treated CNS neurons require new protein and RNA synthesis to undergo an active death programme, and that internucleosomal fragmentation of DNA mediates the etoposide-induced programmed cell death. This culture system of etoposide-treated CNS neurons is thought to be a useful model for the study of programmed neuronal cell death.

Enhancement of Neurite Outgrowth and Aspartate‐Glutamate Uptake Systems in Retinal Explants Cultured with Chick Gizzard Extract

Chicken gizzard extract promoted a long and radially directed neurite outgrowth from retinal explants of 8‐day‐old chick embryo in cultures of 2–3 days. The neurite outgrowth from retinal explants cultured in the absence of gizzard extract was short and restricted to the explant perimeter. The neurite outgrowth promoted by gizzard extract depended strictly on several factors. (a) Fetal calf serum and polycationic substratum were required in this culture system, (b) Pretreatment of the polyornithine‐coated substratum with gizzard extract allowed the retinal explants to extend neurites even in the absence of gizzard extract in the medium. (c) Maximal neurite outgrowth was observed in retinal explants dissected from 8‐day embryos, but thereafter the explants’response to gizzard extract rapidly declined and was almost lost at the 12th day. As a biochemical parameter of differentiation of cultured neuroretina, uptake systems for neurotransmitter candidates were examined in homogenates of retinal explants cultured in the absence or presence of gizzard extract. After 3 days in culture with gizzard extract, the uptake increased for aspartate and glutamate 1.6‐ to 1.8‐fold and for γ‐aminobutyric acid to a lesser degree when examined at a concentration for high‐affinity uptake (10‐6M). In contrast, the uptake capacity for glycine, choline, and dopamine was not altered in explants cultured with or without gizzard extract. Kinetic analysis showed that the enhanced capacity to accumulate aspartate was not due to an alteration of Km, but to an increase of Vmax. The results suggest that one or several factors in chick gizzard muscle promote not only neurite outgrowth but also the aspartate‐glutamate uptake systems in the developing neuroretina, probably related to ganglion cells.

Nerve growth factor and epidermal growth factor rescue PC12 cells from programmed cell death induced by etoposide: Distinct modes of protection against cell death by growth factors and a protein-synthesis inhibitor☆

A rat pheochromocytoma cell line (PC12 cells) died within 24 h in the presence of etoposide (1-40 micrograms/ml), an inhibitor of topoisomerase II. This cytotoxic effect was prevented by either nerve growth (NGF) or epidermal growth factor (EGF). Cycloheximide and actinomycin D also suppressed the cell death as well. Furthermore, a difference among protective modes against etoposide-induced death by growth factors and a protein-synthesis inhibitor was observed: the protective effect of either NGF or EGF remained rather constant as a function of incubation time with etoposide whereas that of cycloheximide declined. These results indicate that etoposide induces programmed death in PC12 cells and that prevention of the programmed cell death by both NGF and EGF is mainly due to inactivation of molecules involved in the death processes rather than suppression of specific protein and/or mRNA synthesis.

Age-dependent survival-promoting activity of vitamin K on cultured CNS neurons

Neurons from the central nervous system (CNS) of rat embryos die within several days when seeded at a low density of 10(4) cells/cm2 and cultured in a serum-free defined medium. Using these culture systems, we searched for agents to promote the survival of these neurons. As a consequence, a fat-soluble vitamin, vitamin K1, was found to possess such kind of activity: more than 50% of the cortical neurons from 19-day-old rat embryos could survive for 4 days in the presence of vitamin K1, whereas almost all neurons died in its absence. The survival-promoting effect of vitamin K1 was found on neurons from not only cortex, but also hippocampus, striatum, and septum. In addition to vitamin K1, vitamin K2 and K3 also showed the same effect on cortical neurons. The effect of vitamins K1 and K2 was observed at concentrations from 10(-8) to 10(-6) M, and that of vitamin K3 was slightly detected at 10(-6) M. Furthermore, we examined the effect on the neurons from 16- and 21-day-old embryos, too. The activity of vitamin K1 was weaker toward the neurons from 21-day-old embryos compared with that toward 19-day-old ones, and was not recognized toward 16-day-old ones. These results suggest the potential role of the K vitamins on the maintenance of the survival of CNS neurons during the later stages of embryogenesis in vivo.

α-Tocopherol supports the survival and neurite extension of neurons cultured from various regions of fetal rat brain

Abstract The effects of α-tocopherol on neurons of fetal rat brain were studied in tissue culture. α-Tocopherol supported the survival and enhanced neurite extension of neurons cultured from cerebral cortex, hippocampus, striatum, and septum. The survival effect was observed at concentrations of 10−9 to 10−6 M. More than 60% of the cortical neurons could survive for 9 days at 10−7 or 10−6 M α-tocopherol, whereas almost all neurons died within 2 days in its absence. These results suggest that α-tocopherol may be involved in mechanisms regulating neuronal survival and neurite formation in the central nervous system, and indicate that α-tocopherol may be an excellent reagent to facilitate neuronal survival in culture.

Interaction of monoclonal antibodies with a neurite outgrowth factor from chicken gizzard extract

A neurite outgrowth factor (NOF), which promotes neurite outgrowth of cultured peripheral neurons, was purified to homogeneity from chicken gizzard extract and used as an immunogen for generating monoclonal antibodies. From 603 hybridoma clones, 55 positive hybridoma cell lines were detected. Four of them (IgG1 (kappa-chain) producers designated as 5-10A, M1-2G, 4-2C and 1-4D) were selected for further characterization. These antibodies immunoprecipitated 125I-labelled gizzard NOF molecules, whereas they did not inhibit the biological activity of NOF (or NOF-like molecules) derived from the tissues tested. The subcellular distribution of NOF in chick embryonal tissues was investigated using monoclonal antibody 4-2C. Both ciliary ganglionic (CG) neurons and gizzard muscle tissues were stained. The NOF content of embryonic CG, measured by an immunoblot assay, increased about 20-fold during embryogenesis from 6 to 15 days, whereas the sensitivity of neurons to NOF reciprocally decreased. At least two different sizes of NOFs (700 and 800 kDa) were found in CG of various ages, with the 800/700 kDa ratio gradually increasing with age. A mixture of these 4 monoclonal antibodies was used for immunoaffinity purification of NOF from chicken gizzard muscles.

Effect of sodium fluorescein on neurite outgrowth from the retinal explant culture: an in vitro model for retinal toxicity

Effect of sodium fluorescein was examined on neurite outgrowth from the retinal explants of chick embryos cultured with chicken gizzard extract, containing macromolecules which promote neurite outgrowth. A dose more than 0.5 mg/ml of fluorescein completely prevented the neurite outgrowth from the retinal explants. The minimum dose of toxic effect of fluorescein on the neurite outgrowth was about 0.2 mg/ml. The fluorescein also caused lysis of pre-existing neurites which had been fully extended by gizzard extract.

Antiserum Against Neurite Outgrowth Factor in Chick Gizzard Extract and Its Inhibitory Effect on Neuritic Response in Cultured Ciliary Neurons

Abstract: Antiserum against a neurite outgrowth factor (NOF) of gizzard extract that promotes neurite outgrowth from dissociated ciliary ganglionic neurons (CG neurons) of 8‐day‐old chick embryo was prepared to determine whether or not the antiserum inhibits neurite outgrowth from cultured neurons or explants of chick and murine tissues. When CG neurons were cultured on a polyornithine‐coated well exposed to NOF (NOF‐bound POR well), marked neurite outgrowth was observed. When NOF‐bound POR wells were exposed to antiserum, neurite outgrowth from CG neurons was gradually inhibited with increasing amounts of antiserum, while exposure to preimmune serum did not prevent neurite outgrowth. Antiserum had no effect on neuronal survival during a 48‐h incubation. The diluted antiserum, which produced nearly 100% inhibition of the NOF activity, was almost equally active in suppressing the activity of NOFs in conditioned media (CM) of various chick embryo tissues, but showed much less inhibitory effects on NOFs in CM of murine tissues. The appearance of neurites from explants of spinal cord, dorsal root ganglion, or retina of chick embryo was also inhibited by the antiserum. These results indicate that antiserum against NOF from gizzard extract suppressed the activity of NOFs from various sources, and that there are species differences in NOFs, at least between chick and murine.

An 82‐Kilodalton Membrane Protein that Inhibits the Activity of Neurite Outgrowth Factor

Abstract: Neurite outgrowth factor (NOF), an extracellular matrix glycoprotein of 700 kilodaltons (kDa), promoted neurite outgrowth from cultured ciliary ganglion (CG) neurons of chicken embryo. A fraction solubilized with Nonidet P‐40 of chicken gizzard muscle membranes inhibited the neurite‐promoting activity of NOF in a dose‐dependent manner, but not that of laminin. Binding of CG neurons to the substratum and their survival were not affected by the extract. The inhibitory activity of the extract was abolished by treatment with trypsin or heat. The molecular size was determined to be about 82 kDa by ligand blotting. The active component was partially purified by column chromatography. It is suggested that this molecule interacts with the domain of NOF responsible for its neurite‐promoting activity and may modulate NOF activity during development in vivo.