Characterization of the retrograde transport of nerve growth factor (NGF) using high specific activity [125I] NGF.

The process of the retrograde transport of nerve growth factor (NGF) has been recharacterized using a high specific activity preparation of[125I]NGF. Most of the general conclusions reached in the previous studies of Hendry, Thoenen and co-workers have been confirmed. However, significant quantitative differences were noted. Intraocular (anterior eye chamber) administration of[125I]NGF (less than 10 ng) resulted in accumulation in the superior cervical ganglia beginning at about 4 h. The ratio of radioactivity in the ipsilateral contralateral ganglia was 15--30:1. Maximal accumulation was seen at about 12h in the hamster and 16 h in rats. This pattern was quite different from that seen in other tissues. The uptake system from the eye of the rat was saturable (half-maximal at 15 ng) with maximal accumulation of 35--40 pg/ganglion. Systemic administration of[125I]NGF (200 ng) to adult rats resulted in no accumulation in SGG or celiac ganglion prior to 3 h, with subsequent rapid accumulation by 6 h and a rapid fall in radioactivity after 12 h. A similar time course was seen in 5-day-old rats, although the time curve was shifted slightly toward shorter time. The radioactivity in ganglia co-migrated with native NGF by SDS gell electrophoresis. Cytochrome c of comparable specific activity was not transported, and NGF did not stimulate the uptake and transport of cytochrome c. The retrograde transport of[125I]NGF was inhibited by the co-administration of biologically active, but not inactive, oxidized derivatives of NGF. By any route of administration, a significant percentage of the transported[125I]NGF was found in a purified nuclear fraction of the ganglia. Coupled with previous observations of specific nuclear NGF receptors in embryonic chick and sympathetic ganglia, this suggests that, after internalization and retrograde transport, NGF may directly act on the nucleus to produce at least some of its effects on the responsive cell.     

Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat

[¹²⁵I]labeled NGF injected in very small quantities into the frontal or dorsal anterior occipital cortex of adult rats, was specifically taken up and transported retrogradely to large, presumably cholinergic neurons in the nucleus basalis region (lateral preoptic nucleus, anterior lateral hypothalamic nucleus, substantia innominata, ventral globus pallidus and internal capsule), as revealed by light microscopic autoradiography. Cells projecting to the injection site in the frontal cortex were localized ipsilaterally in the more caudal parts of the nucleus basalis region, whereas cells projecting to the dorsal anterior occipital cortex could be found throughout the entire extent of the nucleus basalis and also in the vertical and horizontal limb of the nucleus of the diagonal band of Broca. Other nuclei known to project to the cortex (locus coeruleus, substantia nigra, nucleus raphe, thalamus) were consistently found to be unlabeled. In contrast to [¹²⁵I]NGF, injection of [¹²⁵I]cytochrome C failed to label any cell bodies in the basal forebrain nuclei by retrograde transport. This high selectivity for uptake and retrograde transport of NGF indicates the presence of membrane receptors for NGF or a closely related molecule on these cholinergic neurons of the basal forebrain innervating the cerebral cortex.     

The retrograde axonal transport of nerve growth factor

A retrograde axonal transport of nerve growth factor (NGF) from the adrenergic nerve terminals in the mouse iris to the cell bodies of postganglionic sympathetic neurones in the superior cervical ganglion has been demonstrated. After injection of iodinated nerve growth factor (125I-NGF) into the anterior eye-chamber there was a relatively rapid accumulation of radioactivity in the superior cervical ganglia on both injected and non-injected sides, as was the case after subcutaneous injection. However, 4 h after intraocular injection a preferential accumulation of radioactivity became apparent in the superior cervical ganglion on the injected side, and this difference between the ganglia on injected and non-injected sides gradually increased to a maximum at 16 h. Transection of the postganglionic adrenergic fibres as well as the prior intraocular injection of colchicine abolished the preferential accumulation of 125I-NGF in the superior cervical ganglion of the injected side, whereas the destruction of adrenergic nerve terminals by 6-hydroxydopamine did not impair the preferential accumulation. It is concluded that the retrograde axonal transport of NGF, which was estimated to take place at a rate of about 2.5 mm/h, depends on a colchicine-sensitive mechanism as does the orthograde rapid axonal transport. However, the uptake of NGF may not only take place from the nerve terminals but also from the preterminal parts, as has been shown in other studies with horseradish peroxidase. Autoradiographic studies strongly supported the existence of a retrograde transport by showing a clear localization of radioactivity in a small number of neurones in the superior cervical ganglion on the injected side, whereas on the non-injected side there was only a diffuse distribution of radioactivity throughout the ganglion.     

Characterization of antibodies to synthetic nerve growth factor (NGF) and proNGF peptides

Sequence data for the mature nerve growth factor (NGF) protein and its precursor are available from molecular cloning of the NGF gene in several species, including mice, humans, rats, and chickens. Hydrophilicity analysis of the predicted rat and chicken prepro-NGF was carried out to locate putative antigenic determinants. Eight peptides were selected and synthesized based on hydrophilicity profiles. Two peptides represent sequences in the rat (and mouse) pro-NGF, one peptide (our peptide P3) represents a highly conserved region of the mature NGF protein (identical in humans, mice, rats, and chickens), two peptides are specific for the mature chicken NGF, and the remaining three peptides are specific for the mature rat NGF (each with only one amino acid substitution compared with corresponding segments of the mouse NGF). For immunization, the peptides were conjugated to keyhold limpet hemocyanin and used to produce antisera in rabbits. After bleeding, peptide-specific antibodies were purified on affinity columns prepared by coupling each of the synthetic peptides. The different peptide antisera and affinity-purified antibodies then were characterized by enzyme-linked immunoassay (ELISA) and immunohistochemistry of the male mouse submandibular gland, a rich exocrine source of NGF. ELISA analysis showed that all peptide antisera bound two to four orders of magnitude better than normal rabbit serum to a coat of their proper peptide. The higher binding was retained by the purified peptide antibodies compared with normal rabbit immunoglobulin. Specific tests, in which one peptide antiserum was checked against different peptide coats in the ELISA, also showed two to four orders of magnitude higher binding of antibodies to the proper synthetic peptide. The peptide antibodies also were tested for their ability to bind to native mouse beta NGF coated to the immunoplates. Only antibodies raised to the conserved P3 peptide recognized native NGF to an extent similar to that obtained with polyclonal anti-NGF antibodies. Conversely, P3 was well recognized by several different NGF antisera. Immunohistochemically, both peptide antisera against the pro-NGF stained the perinuclear cytoplasm in the basal part of the cells of the granulated convoluted tubules in the mouse submandibular gland.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specificity of the retrograde axonal transport of nerve growth factor

The specificity of the retrograde axonal transport of nerve growth factor (NGF) has been investigated by injecting¹²⁵I-labelled NGF and similar proteins (both with respect to molecular weight and electrical charge at physiological pH) into the anterior eye chamber of adult mice. Previous studies have shown that the difference between the accumulation of radioactivity in the superior cervical ganglion of the injected and non-injected sides is a measure for retrograde axonal transport. Of all the proteins studied (NGF, cytochrome c, insulin, horseradish peroxidase, ovalbumin, bovine albumin, ferritin), NGF was the only which exhibited a statistically significant (2–3 fold) difference between injected and non-injected sides. The specificity of the retrograde transport of NGF was further supported by the finding that relatively small chemical changes, such as oxidation of the tryptophan moieties of the NGF molecule, resulted in a marked reduction of retrograde transport.It is concluded that the system for providing information to the cell body from the adrenergic nerve terminals in the form of NGF is highly specific, and that this specificity does not depend on general physico-chemical properties of the molecule such as size of the molecule and electrical charge at physiological pH. However, the information available so far does not allow one to decide as to whether the selectivity resides in the transport system itself or in the specificity of the uptake mechanism at the nerve terminals.     

The significance of retrograde axonal transport for the accumulation of systemically administered nerve growth factor (NGF) in the rat superior cervical ganglion.

The present study has shown that after intravenous injection of [125I]NGF the time-course of appearance of radioactivity in all organs studied with the exception of sympathetic and sensory ganglia, roughly paralleled that of the blood. The highest levels were reached immediately after injection, after which the radioactivity decayed rapidly within the firsh hour. By contrast, in the superior cervical ganglion there was a small but significant increase within the first hour. After this the radioactivity remained constant for about 4 h and then increased dramatically (7-fold) when the radioactivity in other tissues had declined to very low levels. Measuring the proportion of radioactivity in the plasma which represents immunologically active NGF, we found that within 30 min after injection all the radioactivity represented unchanged [125I]NGF. After this time the proportion of immunologically active NGF decreased gradually and reached a final level of about 10-15%. Evidence that the radioactivity accumulated in the superior cervical ganglion by retrograde axonal transport represents unchanged [125I]NGF was provided by gel electrophoresis. The results are interpreted as follows: the initial small increase in the sympathetic ganglia may result either from [125I]NGF taken up by short collateral fibres within the ganglion or from a direct accumulation of blood-borne [125I]NGF by the cell bodies of the adrenergic neurones. The dramatic increase occurring after 4 h is caused by the moiety of [125I]NGF reaching the cell body by retrograde axonal transport. This interpretation is supported by autoradiographic studies which showed that 1 h after [125I]NGF injection there was only very sparse labelling of the ganglion, whereas 24 h later virtually all the cell bodies were heavily labelled. Moreover, it could be shown that the lag period between intravenous injection and subsequent accumulation of [125I]NGF in the adrenergic cell bodies was considerably shorter after transection of the postganglionic fibres distal to the cell body [the transected fibres were allowed to regenerate for 7 days] resulting in a reduction of the distance between the site of uptake and accumulation.     

The effect of nerve growth factor (NGF) upon axoplasmic transport in sympathetic neurons of the mouse

To study a possible agent which controls axoplasmic transport, the rates of transport were examined in postganglionic sympathetic neurons in adult mice which had been treated with nerve growth factor (NGF). Rates were measured by observing the movement along the nerve of a front of radioactive material after a prior intraganglionic injection ofL-[4,5-³H]leucine. The faster of two rates observed in female mice increased from a control value of1.22 ± 0.48mm/h (N= 15) to an experimental value of 1.83 ± 0.53mm/h (N= 6; P < 0.005)following the intramuscular injection of a total of 34 μg NGF/g body weight. In contrast, the same treatment caused the slower of the two rates to decrease from a control value of2.32 ± 0.81mm/day (N = 12) to an experimental value of 1.63 ± 0.70mm/day (N= 12; P < 0.05). The rate of slow transport was also examined in male animals. Untreated male mice exhibit a lower rate of slow transport (1.08 ± 0.38mm/day (N= 9) than do untreated females of equal age (P < 0.001). The result that nerve growth factor increases the rate of the faster phase of transport, and decreases the rate of the slower phase, indicates that this protein exerts a selective effect on transport.To obtain an independent measure of the influence of NGF, the extent of incorporation ofL-[4,5-³H]leucine into tissue proteins of excised stellate ganglia was assessed after 1 h in organ culture. Animals treated with NGF showed increased incorporation of tritiated leucine. Incorporation of radioactive leucine was correlated with the rateof transport in both control animals (r = 0.61) animals treaetd with NGF (r = 0.67). Within the framework of normal biological function, NGF may exert a controlling influence on both phases of axoplasmic transport in sympathetic postganglionic neurons.     

Nerve growth factor (NGF) in the rat CNS: Absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra

Selective, highly efficient uptake of [125I]NGF by nerve terminals followed by retrograde axonal transport, and specific induction of tyrosine hydroxylase by NGF are well known phenomena in peripheral adrenergic neurons of adult rats. In the present study these parameters were used in order to detect possible interactions of NGF with central catecholaminergic neurons. No selective retrograde transport of [125I]NGF could be detected by light microscopic autoradiography from the caudate nucleus to the dopaminergic neurons in the substantia nigra or from the hippocampus to the noradrenergic nerve cells of the locus coeruleus. Biochemically, no change in tyrosine hydroxylase activity could be observed for up to 3 days after injection of either NGF, anti-NGF antibodies, or control proteins close to the nerve cell bodies in the substantia nigra or the locus coeruleus. These data suggest a fundamental difference between central and peripheral adrenergic neurons with regard to their responsiveness of NGF.     

Reversible sedimentation and masking of nerve growth factor (NGF) antigen by high molecular weight fractions from rat brain

Nerve growth factor (NGF) was recently found to be largely associated with sedimentable fractions of adult rat brain and treatments of the fractions by alkaline pH increased the measurable amount of their NGF antigen as well as its solubilization [M.C. Hoener, E. Hewitt, J.M. Conner, J.W. Costello and S. Varon, Nerve growth factor (NGF) content in adult rat brain tissues is several-fold higher than generally reported and is largely associated with sedimentable fractions, Brain Res., 728 (1996) 47–56; M.C. Hoener and S. Varon, Effects of sodium chloride, Triton X-100, and alkaline pH on the measurable contents and sedimentability of the nerve growth factor (NGF) antigen in adult rat hippocampal tissue extracts, J. Neurosci. Res., in press (1997); C. Zettler, D.C.McL. Bridges, X.-F. Zhou and R.A. Rush, Detection of increased tissue concentrations of nerve growth factor with improved extraction procedure, J. Neurosci. Res., 46 (1996) 581–594]. We have further investigated the reversibility of these pH effects. Reversal of the pH of an adult rat hippocampal tissue extract from 10.5 to 7.4 led to an almost complete transfer of NGF back from nonsedimentable to sedimentable fractions and to a remasking of the previously unmasked portion of NGF antigen. Thus, molecules causing masking and sedimentation of NGF at pH 7.4 were likely to be present in the alkaline extract. A gel filtration column in PBS, pH 10.5 was used to separate such putative binding molecules from the NGF. All of the NGF antigen from rat hippocampal alkaline extract was found to elute with 19 kDa fractions. The same apparent molecular weight was found for mouse submaxillary β-NGF and recombinant human β-NGF. Masking and sedimentation no longer occurred when newly generated 19 kDa rat brain NGF was returned to pH 7.4. When high molecular weight fractions derived from the same gel filtration (in PBS, pH 10.5) were added back to the 19 kDa NGF pool at pH 7.4 and the mixture incubated and centrifuged, the measurability of 19 kDa rat brain NGF antigen was markedly reduced and half of the antigen was recovered in sedimentable fractions. Similar but less dramatic results were obtained when mixing the same high molecular weight fractions with 19 kDa mouse or human β-NGF. These findings provide new opportunities to identify molecules to which NGF may be bound within intact brain tissues.     

Biological importance of retrograde axonal transport of nerve growth factor in adrenergic neurons.

Previous studies have shown that nerve growth factor (NGF) produces a selective induction of tyrosine hydroxylase (TH) in peripheral adrenergic neurons and that NGF is transported retrogradely with a high selectivity from the adrenergic nerve terminals to the perikaryon. In order to investigate the biological importance of retrograde NGF transport, the following experiments have been performed; (a) effect of NGF on TH activity in superior cervical ganglia (SCG) after unilateral injection into the anterior eye chamber and the submaxillary gland; and (b) effect of systemic injection of NGF on TH activity in SCG after blockage of retrograde axonal transport by axotomy. After unilateral injection of NGF into the anterior eye chamber and submaxillary gland of both 8-10-day-old rats and adult mice, the increase in TH activity in the SCG was considerably larger on the injected than on the non-injected side although the adrenergic neurons supplying the two organs do not account for more than 25% of the total number of adrenergic neurons in the SCG. A direct diffusion mechanism could be excluded by the fact that unilateral local injection of [125 I] produced no significant side difference in the accumulation of radioactivity in the SCG 2 after injection whereas after 14 h there was a several-fold difference between the injected and non-injected side. Moreover, the nodose ganglia which are located very close to the SCG exhibited no statistically significant difference in the accumulation of radioactivity at any time. Forty-eight hours after subcutaneous injections of 10 mg/kg of NGF the increase in TH activity of the SCG amounted to 154% on the intact side and to 92% on the axotomized side. However, these experiments do not permit decisions about the extent the axotomy, as such, impaired the response to NGF. It is concluded that the biological effect of NGF results to a considerable extent, from the moiety which reaches the cell body by retrograde transport from the nerve terminals.     

Characteristics of the retrograde axonal transport system for nerve growth factor in the sympathetic nervous system

Retrograde axonal transport of¹²⁵I labeled nerve growth factor (NGF) occurs in the adrenergic neurons of the sympathetic nervous system of rats as well as mice. This transport is saturable, it can be blocked by an excess of unlabeled NGF and shows a specificity for the biologically active form of the protein. Other proteins such as serum albumin, chymotrypsin or insulin are transported in varying, but much smaller, amounts. Although the transport of albumin is blocked by an excess of NGF, the opposite is not true, again suggesting a specific mechanism. NGF antibody, injected subcutaneously, prevents retrograde transport in neonatal but not in more mature animals and the relative resistance of the latter to the action of NGF antiserum may be related to this phenomenon.     

Loss of cell adhesion impairs the early response of TrkA to nerve growth factor (NGF)

Within the in vitro model of nerve growth factor (NGF)-dependent differentiation of PC12 cells, loss of adhesion is known to interfere with certain neurotrophic responses and not with others. The present analysis of early NGF signaling shows that the upstream activation of TrkA, as well as the recruitment of the adaptor protein Shc, are dramatically attenuated upon loss of cell adhesion, while the downstream activation of ERK1/2 is unaffected. That loss of cell adhesion interferes with the primary signaling response to NGF (i.e. autophosphorylation of TrkA) may explain why suspended PC12 cells fail to morphologically differentiate in response to NGF. The possibility that established adhesion-driven mechanisms underlie the full responsiveness of TrkA to NGF is discussed.     

Retrograde axonal transport of [125I]nerve growth factor in ileal mesenteric nerves in vitro: effect of streptozotocin diabetes

The retrograde axonal transport of intravenously administered [125I]nerve growth factor ([125I]NGF) was examined in ileal mesenteric nerves maintained for short periods in vitro. [125I]NGF was injected systemically, and at various times thereafter mesenteric pedicles were ligated and incubated in vitro in Krebs-Henseleit medium under a number of different conditions. Retrogradely transported [125I]NGF began to accumulate distal to the ligature after an initial lag period and increased in a linear fashion for 3-4 h. The amount of retrogradely transported [125I]NGF was proportional to the length of the ileum innervated by each pedicle, which allowed for comparison of ileal segments of different lengths. Retrograde axonal transport of [125I]NGF was inhibited by vinblastine, colchicine and incubation in the cold, and was decreased by agents that interfere with oxidative or glycolytic metabolism. The accumulation of retrogradely transported [125I]NGF in ileal mesenteric nerves of 1-9 day streptozotocin diabetic animals placed in an in vitro bath containing normal (5.5 mM) glucose was decreased 40% compared to control animals. The induction of diabetes in vivo resulted in a greater decrease in the early phases of [125I]NGF export from ileal mesenteric nerve terminals compared to later phases. Ileal mesenteric nerve segments derived from untreated controls were incubated in vitro in media containing increased concentrations of glucose (27.5 and 50 mM) without reproducing the NGF transport defect found in diabetic animals.     

The effects of nerve growth factor (NGF) and antiserum to NGF on the development of embryonic sympathetic neurons in vivo

The role of nerve growth factor (NGF) in the development of embryonic sympathetic neurons was examined in vivo. Individual mouse embryos received transuterine injections of NGF or antiserum to NGF (anti-NGF), and the effects on the superior cervical ganglion (SCG) were studied. Treatment with NGF at any gestational stage, from the time of ganglion aggregation to birth, increased ganglion tyrosine hydroxylase (T-OH) activity. Both the number of catecholaminergic neurons and T-OH activity per neutron were increased. Choline acetyltransferase (ChAc) activity was increased by NGF at early gestational stages, but not at later stages. These observations suggest that perikarya containing ChAc are responsive to NGF, whereas preganglionic nerve terminals are not. Treatment with anti-NGF rapidly and permanently decreased ganglion T-OH activity. The effects of anti-NGF were more pronounced at later gestational stages, suggesting that ganglia become increasingly dependent on NGF during development. Alteration of maternal levels of NGF had no effect on development of the embryonic SCG, suggesting that local embryonic concentrations of NGF are responsible for modulating sympathetic ontogeny.