Cholinergic Neuronotrophic Factors. Concurrent Activities on Certain Nerve Growth Factor-Responsive Neurons

Abstract: We had previously reported that in vitro survival of chick embryo ciliary ganglionic neurons can be assured by the addition to the culture medium of appropriate amounts of soluble macromolecular agents termed ciliary neuronotrophic factors. Particularly rich sources of one such factor are aqueous extracts from chick embryo intraocular tissues that include the smooth and striated musculature innervated by ciliary ganglionic neurons. We report here that this eye extract also contains agents that we term ganglionic neuronotrophic factors that support the survival of 11-day chick embryo sympathetic and neonatal mouse dorsal root ganglionic neurons, two traditional targets of nerve growth factor (NGF). Using a recently developed microassay procedure we found that these ganglionic activities are not inactivated by rabbit, rat, or guinea pig antisera raised against the 2.5S (beta) subunit of male mouse submaxillary NGF, rabbit antisera against 7S NGF, or quail antisera against cobra venom NGF. Both the ciliary and ganglionic activities can be quantitated simultaneously by using 24-h in vitro microassays, thus permitting a direct comparison of their respective properties. Both activities were found to (a) adsorb to DE52 cellulose and coelute at a similar salt concentration, (b) focus and be recovered from isoelectric polyacrylamide gels at exactly the same pH region, (c) be heat-and partially acid-labile, but base-stable, and (d) be inactivated by exposure to trypsin. These results suggest that the ciliary and ganglionic neuronotrophic activities are associated with the same protein.     

Cholinergic Neuronotrophic Factors: Fractionation Properties of an Extract from Selected Chick Embryonic Eye Tissues

Abstract: An aqueous extract derived from selected intraocular tissues of 15-day chick embryos contains a soluble macromolecular agent which is capable of ensuring the survival of 8-day chick embryonic ciliary ganglionic neurons in monolayer culture. When this ciliary neuronotrophic factor (CNTF) was concentrated using ultrafiltration and subjected to Sephadex G100 and G200 chromatography, activity was detected in most of the eluted fractions. A peak of the most active fractions was eluted in a region corresponding to a molecular weight of 35–40 ± 10³ and contained about 20-30% of the applied protein. CNTF activity bound readily to DE-52 cellulose resin at neutral pH and was eluted with NaCl in a narrow region containing about 20-40% of the applied protein. Gel electrophoretic staining profiles of the active DE52 fraction indicated considerable (but still only partial) simplification in protein composition. While significant CNTF activity losses were incurred in response to each of the above treatments, an active material could be conveniently generated in one working day in milligram amounts having a specific activity of 60,000 trophic units/mg protein. This trophic activity is in the same range as that of the only other known neuronotrophic factor, Nerve Growth Factor.     

Cholinergic neuronotrophic factors: VI. Age-dependent requirements by chick embryo ciliary ganglionic neurons

During development, ciliary ganglionic neurons become postmitotic and extend neurites in apparent independence of the presence of their future intraocular innervation targets. After reaching their peripheral innervation territory, however, these neurons become target dependent and about half of them die. We have previously reported that chick embryo intraocular target tissues contain a ciliary neuronotrophic factor (CNTF), which can be extracted and partially purified in a soluble form and which ensures near-total survival of 8-day chick embryo ciliary ganglionic neurons in monolayer cultures. In this study we have dissociated and cultured ciliary ganglia from embryonic Day (ED) 5 through 14, and examined dependence and responsiveness of their neurons to exogenously added CNTF. Two cell classes (dark and bright) could be distinguished by phase microscopy and differentially counted in cell dissociates from ED7–14, but not in ED5–6 ones. Dark cell number per ganglion increased from 6000 to 78,000 over this developmental time period. In contrast, bright cells (putative neurons) declined from a maximum of about 10,000 to 6000, suggesting a correlation with the expected neuronal cell death in vivo. Dissociated cells from ED5–14 ganglia were seeded on a polyornithine substratum coated with neurite promoting factor, cultured for 24 hr with or without added CNTF, and numerically examined for survival and neuritic development. Cultures from ED7–14 ganglia showed two cell categories: (i) flat nonneuronal elements dramatically increased in number with ganglionic age (thereby correlating with the increasing number of dark cells in the dissociates) and (ii) large, bright cells (often displaying neurite outgrowth) decreased in number in parallel with bright cell number in the dissociate. The survival of these neuronal elements was strictly dependent on exogenously added CNTF between ED7 and 10, but became progressively independent with older ages. ED14 neurons (fully capable of surviving for 24 hr without added CNTF) continued to require CNTF for neurite extension, thus displaying retained sensitivity to this factor. Although the ED5–6 cultures contained well-recognizable flat cells, the dominant category comprised cells with variable morphology, practically all of which exhibited neurite-like processes. Both the survival and neurite extension of these cells, which we tentatively interpret as immature neurons were independent of the presence of added CNTF.     

Purified proteins acting on cultured chick embryo ciliary ganglion neurons

Chick embryo ciliary ganglion neurons in dissociated monolayer culture have been used to examine molecular requirements for neuronal survival and neurite growth. These neurons will rapidly die in vitro unless supplied with an adequate level of ciliary neuronotrophic factor (CNTF), and even in the presence of CNTF they will not vigorously extend neurites on polyornithine substrata unless supplied with appropriate amounts of polyornithine-binding neurite-promoting factors (PNPFs). Recent work on the purification and partial characterization of embryonic chick eye CNTF and rat schwannoma PNPF is reviewed, and in vitro responses of ciliary ganglion neurons to other purified proteins such as laminin, fibronectin, insulin, and nerve growth factor are mentioned.     

Differential Effects of Nerve Growth Factor and Ciliary Neuronotrophic Factor on Catecholamine Storage and Catecholamine Synthesizing Enzymes of Cultured Rat Chromaffin Cells

The effects of nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF) on catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were studied in adrenal chromaffin cells cultured from 8-day-old rats. Both NGF and CNTF enhanced chromaffin cell survival and partially prevented losses of adrenaline during the 4-day culture period in a dose-dependent manner. CNTF was more potent, although cellular levels of adrenaline and noradrenaline were not maintained. NGF did not add to the effect of CNTF. The effect of CNTF on catecholamine storage was not accompanied by changes in the activities of TH and PNMT. In contrast, NGF induced TH but not PNMT activity. These data indicate differences between the mechanisms by which NGF and CNTF affect adrenal chromaffin cells.     

A ciliary neuronotrophic factor from peripheral nerve and smooth muscle which is not retrogradely transported

We have found that a CNTF-like molecule which supports ciliary and sympathetic neurons is not retrogradely transported in either sympathetic or parasympathetic nerves. The factor has an apparent M_r of 21 kDa, a pI of 4.9, and is present in peripheral nerves and smooth muscle of the chick. Our experiments indicate that CNTF-like activity does not accumulate on the distal side of ligated chickexpansor nerves. In contrast, there is a clear accumulation of NGF. The activity further differs from NGF in that it is not removed from a smooth muscle of the chick wing by innervating sympathetic fibers. Transection of these fibers does not lead to an accumulation of ciliary activity in theexpansor secundariorum muscle, suggesting that neurons do not actively deplete the muscle of factor by retrograde transport. Finally, recombinant CNTF or semi-purified preparations of CNTF-like activity labelled with¹²⁵I were not transported to the ciliary ganglion of chicks following injection of biologically active material into the eye. Our results suggest either that endogenous CNTF does not act as a survival factorin vivo, or that retrograde transport is not a property inherent to all neuronotrophic molecules.Special issue dedicated to Dr. Lawrence Austin     

Characterization and Partial Purification of a Novel Neuronotrophic Factor from Bovine Seminal Vesicle

Extracts from bovine seminal vesicles have been shown to contain high concentrations of nerve growth factor (NGF)-like biological activity and of the NGF protein with properties corresponding to that of NGF from other sources. We now demonstrate that a second neuronotrophic protein, termed seminal vesicle-derived neuronotrophic factor (SVNF), is present in seminal vesicle extracts (SVEs), which could not be distinguished from NGF on the basis of biological activity. SVNF has neuronotrophic activity on NGF target cells like embryonic chicken-sensory and sympathetic neurons, sympathetic neurons, and chromaffin cells from neonatal rats, but it is inactive on embryonic chicken ciliary or neonatal rat nodose ganglion neurons. It also stimulates fiber outgrowth from rat pheochromocytoma (PC 12) cells. In gel filtration chromatography on Biogel A 1.5 m, the activity is eluted with an apparent molecular weight of 40 kilodaltons, and by preparative isoelectric focusing, the isoelectric point was determined to be in the neutral range (6.8-7.8). The biological activity of SVNF, in contrast to that of NGF, is partially retained after preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and can be electrophoretically eluted with an apparent molecular weight of 16-20 kilodaltons. Electrophoretically purified SVNF is not inhibited by antisera to mouse NGF, but its activity is increased greater than 10-fold in the presence of very low concentrations of NGF. For partially purified SVNF, a specific activity of 2.9-5.8 X 10(5) biological units/mg of protein was determined in the presence of subthreshold NGF concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ciliary Neuronotrophic Factor Stimulates Choline Acetyltransferase Activity in Cultured Chicken Retina Neurons

It has been demonstrated that cultured cholinergic retinal neurons from 8-day-old chicken embryos respond to a polypeptide factor present in retinal cell-conditioned medium (RCM) and in retinal extracts. Compared with control cultures, the activity of acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; ChAT) is enhanced more than twofold in neuronal retinal cultures grown for 7 days in the presence of RCM. The present study demonstrates that both ciliary neuronotrophic factor (CNTF), which is characterized by its trophic activity on parasympathetic ciliary neurons, and RCM exhibit identical stimulatory effects on ChAT activity in retinal monolayer cultures. Similarly, RCM supports the in vitro survival of ciliary neurons to the same extent as CNTF. The active species in RCM has a molecular weight (20,900 +/- 1,000) identical to that of CNTF, as determined by preparative sodium dodecyl sulfate gel electrophoresis. The results indicate that cholinergic retinal neurons represent a central neuronal target for CNTF or a closely related protein.     

Neuronal survival in intact ciliary ganglia in vivo and in vitro: Ciliary neuronotrophic factor as a target surrogate

Ciliary neuronotrophic factor (CNTF) requirements for neuronal survival in the intact ciliary ganglion (CG) have been investigated in organ culture. Exogenous CNTF was not essential for neuronal survival until embryonic Day 8. Three-day cultures from 5-day ganglia were similar with or without CNTF, showing numerous neurons and extensive neuritic development. In 3-day cultures from 8-day-old ganglia, however, no neurons survived without CNTF, and the ganglia contained only nonneuronal cells and cell debris. Similar ganglia cultured with CNTF contained many neurons, surrounded by nonneuronal cells, and abundant neuritic processes. Morphologic maturation of the neurons was less advanced in CNTF-supported ganglia than in their in vivo counterparts.     

Preparation and biological properties of native and recombinant ciliary neurotrophic factor

CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE). In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons. The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed. With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves. After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons. In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons. © 1992 John Wiley & Sons, Inc.