The response of cultured trigeminal and spinal cord motoneurons to nerve growth factor

Dissociated neurons from the trigeminal (V) region of the metencephalic basal plate or the ventral spinal cord from chick embryos of Day 4 (V basal plate) or Day 5 (spinal cord) were cultured on a laminin substratum either in the presence of nerve growth factor (NGF) or in control medium. Assessment was made of neuronal survival, the amount of neurite elaborated, and the percentage of neurons initiating neurites. The presence of motoneurons was verified by retrograde labeling with the fluorescent dye diI. NGF was found to significantly increase the quantity of neuritic processes produced by the spinal cord dissociates at both 24 and 48 hr in vitro. The percentage of neurons initiating neuritic processes was significantly increased by NGF in the trigeminal population at 48 hr in vitro. Neuronal survival was not enhanced by NGF in either group. Both trigeminal and spinal cord neurons were also found to specifically bind 125I-NGF in culture. These results provide direct evidence for an influence of NGF on process formation of early embryonic motoneurons in culture.     

The effects of nerve growth factor and dibutyryl cyclic AMP on cytoskeletal densities in cultured sensory ganglia.

The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths. As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in vitro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofilaments was seen at 24 hr, with equal densities of both cytoskeletal elements present after 48 hr in vitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC, implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neurites of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.     

Stage-dependent stimulation of neurite outgrowth exerted by nerve growth factor and chick heart in cultured embryonic ganglia

The ability of embryonic chick heart to elicit neuritic outgrowth in different ganglia was tested to examine (1) whether stimulative activity is possessed by the heart only at specific stages and (2) whether the ability of the ganglionic neurons to respond is limited to certain periods of development. As an assay, ganglia were explanted into thin collagen gels with ventricular tissue placed at a distance of about 1 mm. Neuritic outgrowth was measured after 2 days. Control ganglia and ganglia cultured with added nerve growth factor (NGF) were also scored. Four types of tested ganglia, including the ciliary ganglion, showed a peak in neuritic outgrowth when cultured with heart of embryonic Day 18, at about which age the heart becomes sympathetically innervated in ovo. No age-related size differences that could account for this temporal pattern were found among the heart explants when measuring their protein content. A peak in neuronal susceptibility to heart tissue was evident in the 6-day ciliary ganglion and in the 8-day paravertebral, Remak, and spinal ganglia, roughly coinciding with the onset of fibre outgrowth in ovo. Neurite extension is concluded to have been triggered by a factor spread from the heart explants and being distinct from the mouse type of NGF since anti-NGF did not at any stage block the events and since added NGF at all stages failed to evoke neurite formation in the ciliary ganglia. A testable hypothesis is that this factor regulates the growth of sympathetic and possibly parasympathetic and sensory fibres in the developing chick heart.     

Metabolic requirements for differentiation of embryonic sympathetic ganglia cultured in the absence of exogenous nerve growth factor.

The embryonic mouse superior cervical ganglion (SCG) in culture was employed to define the role of ongoing metabolic processes in morphological and biochemical development. The 14 gestational day SCG does not require added nerve growth factor (NGF) for differentiation in vitro. Consequently, its use allows study of intraganglionic regulation of neuronal growth in the absence of complicating, exogenous growth factors. Ganglia were cultured without added NGF, in medium containing various metabolic inhibitors; neurite elaboration and development of tyrosine hydroxylase (T-OH) activity, a biochemical marker of adrenergic maturation, were evaluated. Neurite elaboration proceeded normally with inhibition of RNA synthesis by actinomycin D, or of protein synthesis by cycloheximide or puromycin. In contrast, inhibition of RNA or protein synthesis prevented normal development of T-OH activity. However, neurites and T-OH developed normally in the presence of DNA synthesis inhibition by cytosine arabinoside, which markedly reduced the nonneuronal cell population. These observations suggest that neurite elaboration and the ontogenetic increase in T-OH activity are regulated differently in ganglia cultured in the absence of exogenous NGF. Moreover, the initial outgrowth of neurites and increase in T-OH activity may occur independent of peripherally migrating support cells in this embryonic ganglion.     

Effect of keratinocyte growth factor on cell viability in primary cultured human prostate cancer stromal cells

In normal prostate, keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7) serves as a paracrine growth factor synthesized in stromal cells that acts on epithelial cells through its receptor, KGFR. KGF and KGFR were found in human cancer epithelial cells as well as stromal cells. Since KGF expressed in epithelial cells of benign prostatic hyperplasia (BPH) and in prostate cancer, it has been suggested that KGF might act as an autocrine factor in BPH and prostate cancer. To investigate the roles of KGF in cancerous stroma, primary cultured human prostate cancer stromal cells (PCSCs) were isolated and evaluated. These PCSCs possessed estrogen receptors and KGFR, but not androgen receptor as determined by RT-PCR and Western blot, respectively. KGF exhibited mitogenic and anti-apoptotic effects that correlated with induction of cyclin-D1, Bcl-2, Bcl-xL and phospho-Akt expression in PCSCs, where treatment with KGF antiserum abolished cell proliferation and anti-apoptotic protein expression. PCSCs exposed to KGF for various time periods resulted in phosphorylation of Akt and subsequent up-regulation of Bcl-2. KGF modulated dynamic protein expression indicated that KGF triggered cell cycle machinery and then activated anti-apoptotic actions in PCSCs. Cell proliferation analysis indicated that tamoxifen or ICI 182,780 reduced cell viability in a dose-dependent manner; however, KGF prevented this inhibition, which further demonstrated KGF triggered anti-apoptotic machinery through activating Bcl-2 and phospho-Akt expression. In summary, KGF has an autocrine effect and serves as a survival factor in primary cultured human prostate cancer stromal cells.     

Sterol-like compound from sensory ganglia. Effect of a nerve growth factor and insulin on its biosynthesis

1. Sensory ganglia from 8-day-old chick embryos were incubated with a specific nerve growth factor and with insulin. 2. From the total lipid extract of the ganglia a compound with steroid characteristics was isolated. 3. The synthesis of this compound, measured spectrophotometrically, diminished after addition of the nerve growth factor and insulin to the incubation medium. 4. The incorporation of sodium [2-(14)C]acetate and dl-[2-(14)C]mevalonic acid into total lipids of the sensory ganglia was stimulated by the nerve growth factor and insulin, but the radioactivity of the sterol-like compound was slightly lower. The incorporation of labelled mevalonic acid either into total lipids or into the sterol-like compound was about 25% lower. 5. About 20% of the acetate incorporated into total lipids and about 87% of the mevalonic acid were recovered in the sterol-like compound.     

Appearance of nerve growth factor receptors on cultured neural crest cells

Light microscopic radioautography of differentiating quail neural crest cultures (1 to 2 weeks after explanation) incubated with Iodine-125-labeled nerve growth factor (125I-NGF) revealed that approximately 35% of the cells bound NGF. The binding was specific and saturable; it was blocked by an excess of nonradioactive NGF, and was not detected following incubation with biologically inactive 125I-NGF. In addition, the binding did not appear to be blocked or diminished by insulin. Cell cultures prepared from somites or notochord showed no specific binding of 125I-NGF. Melanocytes comprised approximately 10% of the cell population in these cultures and appeared to be unlabeled. The subpopulation of cells with NGF receptors that were morphologically similar to other non-melanocyte unlabeled cells present in the neural crest cultures are probably the targets of the factor during differentiation and development. In contrast, there was no evidence of 125I-NGF binding by premigratory neural crest (adherent to the isolated neural tube) or by early migratory neural crest cells (24 hr after explantation). Both of these types of neural crest cells are relatively undifferentiated. The cells of the neural tube were also unlabeled. The binding of 125I-NGF to differentiating neural crest cells was not noticeably diminished by a brief pretreatment with trypsin or Dispase, enzymes used in the isolation of neural tubes. Hence, the absence of NGF receptors on premigratory neural crest and early migratory neural crest cultures was not due to enzymatic alterations of the receptor. It seems, therefore, that receptors for NGF appear on neural crest cells during the time when these cells are acquiring their phenotypic characteristics.     

Physical properties of the detergent-extracted nerve growth factor receptor of sympathetic ganglia.

The nerve growth factor (NGF) receptor from microsomes of adult rabbit superior cervical ganglia has been solubilized with Triton X-100 and sodium deoxycholate. The physical properties of the detergent-extracted NGF receptor were assessed by Sepharose 6B chromatography and sucrose density gradient ultracentrifugation studies in H2O and D2O. The predominant form of the NGF receptor has a Stokes radius of 71 A, a partial specific volume of 0.74 ml/g, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.8. From these parameters, it can be calculated that the NGF receptor in Triton X-100 is a minimally hydrophobic, highly asymmetric, intrinsic membrane protein with a molecular weight of approximately 135,000. A form of the receptor with a sedimentation coefficient of 10.4 S was occasionally seen which appears to represent an aggregated form of the 4.3 S moiety.     

Loss of nerve growth factor receptors in sympathetic ganglia from aged mice

[125I]-Nerve growth factor (NGF) binding was studied in superior cervical ganglia from mice 4-7 months and 24-27 months of age. Scatchard analysis demonstrated losses of both high and low affinity components of NGF receptor. These results indicate loss of NGF receptors may lead to the diminished responsiveness to NGF in aged sympathetic ganglion neurons.     

Ceramide inhibits axonal growth and nerve growth factor uptake without compromising the viability of sympathetic neurons.

Ceramide inhibits axonal growth of cultured rat sympathetic neurons when the ceramide content of distal axons, but not cell bodies, is increased (Posse de Chaves, E. I., Bussiere, M. Vance, D. E., Campenot, R. B., and Vance, J.E. (1997) J. Biol. Chem. 272, 3028-3035). We now report that inhibition of growth does not result from cell death since although ceramide is a known apoptotic agent, C(6)-ceramide given to the neurons for 24 h did not cause cell death but instead protected the neurons from death induced by deprivation of nerve growth factor (NGF). We also find that a pool of ceramide generated from sphingomyelin in distal axons, but not cell bodies, inhibits axonal growth. Analysis of endogenous sphingomyelinase activities demonstrated that distal axons are rich in neutral sphingomyelinase activity but contain almost no acidic sphingomyelinase, which is concentrated in cell bodies/proximal axons. Together, these observations are consistent with the idea that generation of ceramide from sphingomyelin by a neutral sphingomyelinase in axons inhibits axonal growth. Furthermore, we demonstrate that treatment of distal axons with ceramide inhibits the uptake of NGF and low density lipoproteins by distal axons by approximately 70 and 40%, respectively, suggesting that the inhibition of axonal growth by ceramide might be due, at least in part, to impaired endocytosis of NGF. However, inhibition of endocytosis of NGF by ceramide could not be ascribed to decreased phosphorylation of TrkA.     

Developmental changes of nerve growth factor levels in sympathetic ganglia and their target organs

The predominant source of nerve growth factor (NGF) used by mature sympathetic neurons originates in their target organs (Heumann, R., Korsching, S., Scott, J., and Thoenen, H. (1984), EMBO J. 3, 3183-3189; Korsching, S., and Thoenen, H. (1985), J. Neurosci. 5, 1058-1061). We have determined the NGF content of two sympathetically innervated mouse organs, submandibular gland and heart ventricle, and of sympathetic ganglia from mouse and rat between embryonic Day 12 (E12) and adulthood. NGF levels were measured by a two-site enzyme immunassay (Korsching, S., and Thoenen, H. (1983), Proc. Natl. Acad. Sci. USA 80, 3513-3516). In heart ventricle and submandibular gland, NGF first became detectable around the time of initial innervation by sympathetic neurons (E12 and E13, respectively) and increased respectively 14- and 7-fold in the following 2 days, to reach adult levels already at E14 for heart ventricle (1.4 +/- 0.2 ng NGF/g wet wt). NGF in the superior cervical ganglion (SCG) was first detected at the same time as in its target organ, the submandibular gland. NGF content in the SCG then increased 6-fold during the next 2 days and continued to increase until the end of the third postnatal week, when adult levels were reached. Although the levels of NGF in the adult mouse submandibular gland are sexually dimorphic and six orders of magnitude higher than those in other sympathetic target organs, no sex difference in the NGF content was found in either developing submandibular gland or SCG until the end of the third postnatal week. Moreover, the steep NGF increase observed in the male submandibular gland after postnatal Day 18 (250-fold within the following 3 days and up to the 55,000-fold in the next 7 days) was not reflected in a corresponding increase in the NGF content of the male SCG. These data indicate that, in accordance with earlier findings (see Levi-Montalcini, R., and Angeletti, P. U. (1968), Physiol. Rev. 48, 534-569), SCG neurons do not have access to the large amounts of NGF synthesized during and after adolescence in the mouse submandibular gland. Our results support the concept that initial fiber outgrowth of sympathetic neurons is neither dependent on NGF nor mediated by it. The time course of NGF levels in the SCG is consistent with the concept that sympathetic neurons are provided with NGF by means of retrograde axonal transport from the innervated organs already early in development.