Involvement of ras p21 in neurotrophin-induced response of sensory, but not sympathetic neurons

Little is known about the signal transduction mechanisms involved in the response to neurotrophins and other neurotrophic factors in neurons, beyond the activation of the tyrosine kinase activity of the neurotrophin receptors belonging to the trk family. We have previously shown that the introduction of the oncogene product ras p21 into the cytoplasm of chick embryonic neurons can reproduce the survival and neurite-outgrowth promoting effects of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and of ciliary neurotrophic factor (CNTF). To assess the potential signal- transducing role of endogenous ras p21, we introduced function-blocking anti-ras antibodies or their Fab fragments into cultured chick embryonic neurons. The BDNF-induced neurite outgrowth in E12 nodose ganglion neurons was reduced to below control levels, and the NGF- induced survival of E9 dorsal root ganglion (DRG) neurons was inhibited in a specific and dose-dependent fashion. Both effects could be reversed by saturating the epitope-binding sites with biologically inactive ras p21 before microinjection. Surprisingly, ras p21 did not promote the survival of NGF-dependent E12 chick sympathetic neurons, and the NGF-induced survival in these cells was not inhibited by the Fab-fragments. The survival effect of CNTF on ras-responsive ciliary neurons could not be blocked by anti-ras Fab fragments. These results indicate an involvement of ras p21 in the signal transduction of neurotrophic factors in sensory, but not sympathetic or ciliary neurons, pointing to the existence of different signaling pathways not only in CNTF-responsive, but also in neurotrophin-responsive neuronal populations.     

Time-Resolved Signaling Pathways of Nerve Growth Factor Diverge Downstream of the p140trk Receptor Activation Between Chick Sympathetic and Dorsal Root Ganglion Sensory Neurons

Abstract: We have recently shown that the small GTP binding protein p21 ras is essential for nerve growth factor (NGF)-mediated survival of peripheral embryonic chick dorsal root ganglia (DRG) sensory but not sympathetic neurons. To investigate at which level of the signaling cascade the pathways diverge, we have studied the time-resolved pattern of NGF-stimulated tyrosine phosphorylation of proteins within 4 h after addition of the neurotrophin. In both chick sympathetic neurons [embryonic day (E) 12] and DRG sensory neurons (E9) NGF induces within 1 min the autophosphorylation of the receptor tyrosine kinase p140trk. However, the pattern of substrate protein tyrosine phosphorylation downstream of p140trk is distinctly different in both neuronal subtypes. In sympathetic neurons, we observe within 1 min the tyrosine phosphorylation of a new substrate protein, p105, reaching maximal levels at 3 min. Tyrosine phosphorylation of p105 remains elevated for up to 4 h. Subsequent to p105, NGF induces the tyrosine phosphorylation of p42, a protein belonging to the family of mitogen-activated protein (MAP) kinases. This stimulation is transient, reaching maximal levels at 10 min and returning to very low levels already after 2 h. In DRG sensory neurons, tyrosine phosphorylation of p105 is weak and very short lived, disappearing already after treatment with NGF for 10 min. In contrast, activation of MAP kinase p42 in DRG sensory neurons is more stable than in sympathetic neurons. All NGF-stimulated tyrosine phosphorylation events were inhibited by preincubation of neurons with the tropomyosin-related kinase (trk) inhibitor K252a. We suggest the working hypothesis that persistent tyrosine phosphorylation of p105 may play a role in the p21ras-independent NGF survival pathway of chick sympathetic neurons.     

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

The survival of early chick sympathetic neurons in vitro is dependent on a suitable substrate but independent of NGF

The neuronal cell population of lumbosacral sympathetic ganglia from 7-day-old chick embryos is characterized by a high proportion of cells with the ability to proliferate in culture (Rohrer and Thoenen, 1987). It is now demonstrated that neither proliferation nor survival of these neurons depend on the presence of nerve growth factor (NGF). However, neuronal survival did depend on the culture substrate used: on laminin, E7 neurons survived and their number increased due to proliferation, whereas on fibronectin (FN) or a substrate of molecules from heart cell-conditioned medium (HCM) a significant number of the cells died during early culture periods. Less than 70 and 50% of the number of neurons surviving on a laminin substrate were found on FN and HCM, respectively, after 3 days in culture. Although NGF did not affect neuronal survival, a small increase in neurite extension on these substrates was observed in the presence of NGF. Furthermore, although NGF did not prevent neuronal death after extended culture periods, this could be prevented by elevated extracellular potassium concentrations. Sympathetic neurons of E8 chick embryos however showed a strikingly different response to NGF compared with those of E7: whereas neuronal survival on laminin was not influenced by NGF, a significant effect of NGF on survival and on neurite extension was observed for E8 neurons on a HCM substrate. In contrast to cells from E7 and E8 embryos, the majority of neurons from E11 chick embryos required NGF for survival even on a laminin substrate as described previously (D. Edgar, R. Timpl, and H. Thoenen, 1984, EMBO J. 3, 1463-1468). These results demonstrate that while sympathetic neurons from E7 chick embryos do not depend on the soluble neurotrophic factor NGF for survival in vitro, they are dependent on molecules of the extracellular matrix. With increasing age, the survival requirements demonstrated in vitro change toward the classical pattern of NGF dependency. Low amounts of laminin-like immunoreactivity were shown to be present in sympathetic ganglia of E7 chick embryos which were then shown to increase as development proceeded. These data indicate that laminin may play a role in the survival and development of chick sympathetic neurons not only in vitro, but also in vivo.     

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.     

Promotion of survival and neurite outgrowth of cultured peripheral neurons by exogenous lipids and detergents

Gangliosides, in particular the monosialoglycosphingolipids Gtet 1 (GM1), have previously been implicated in the mediation of neuronal rescue and restitutional axonal growth, both in vitro and subsequent to brain and peripheral nerve lesions. In the present study it is shown that the bis-sialosyl gangliosides Gtet2b and Gtet3b, but not the gangliosides Gtet2a and Gtet1, promote the survival of dissociated dorsal root ganglion (DRG) neurons cultured from Embryonic Day (E) 8 chicks (DRG8) almost to the same extent as nerve growth factor (NGF). Ciliary ganglion (CG) neurons from E8 chicks (CG8) and DRG10 neurons were virtually not supported suggesting considerable specificity in terms of neuronal targets and developmental stages being addressed. Moreover, a variety of other lipids including cerebroside (Cb), dipalmitoylphosphatidylcholine (DPPC) and -serine (DPPS), sulfatide (Sf), and sphingomyelin (Sm) were tested for putative survival promoting activity toward chick CG, DRG, and lumbar sympathetic ganglion (SG11) neurons. At the highest concentration employed (2.5 x 10(-5) M), Sm, DPPC, and DPPS maintained between 45 and 65% of the plateau survival with CG8 (maximally supported by ciliary neuronotrophic factor (CNTF], DRG8, and DRG10 neurons, and 30 to 40% with SG11 neurons. Cb supported CG8 neurons at about 55% of the plateau value achieved with CNTF, but had hardly any effect on the other neuron populations tested. Control experiments using highly enriched neurons and serum-free conditions assured that the effects were unlikely to be mediated by serum components or nonneuronal cells. A variety of detergents, in particular Triton X-100, also promoted the survival of CG8 and DRG10 neurons. Ganglioside Gtet1, Sm, and Triton X-100 shifted the NGF titration curve for DRG10 neurons between 6- and 15-fold in a dose-dependent manner suggesting synergisms between NGF and lipids for neuronal maintenance. These results document the neuronotrophic potency of certain gangliosides, a heterogeneous group of structurally unrelated lipids, and detergents. The mechanisms by which these agents modulate neuronal survival still await clarification.     

Sympathetic neuronal survival induced by retinal trophic factors

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti‐NGF (1 μg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1–50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin‐receptors TrkA, TrkB, or TrkC had no effect on RCM‐induced sympathetic neuron survival. The survival effects were not blocked by anti‐GDNF, anti‐TGFβ, and anti‐CNTF and were not mimicked by FGFb (0.1–10 nM). LY294002 at 50 μM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high‐molecular weight retinal secreted factor that supports sympathetic neurons in culture. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 13–23, 2002     

Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia

The influence of the neural tube on early development of neural crest cells into sensory ganglia was studied in the chick embryo. Silastic membranes were implanted between the neural tube and the somites in 30-somite-stage embryos at the level of somites 21-24, thus separating the early migrated population of neural crest cells from the neural tube. Neural crest cells and peripheral ganglia were visualized by immunofluorescence using the HNK-1 monoclonal antibody and several histochemical techniques. Separation of crest cells from the neural tube caused the selective death of the neural crest cells from which dorsal root ganglia (DRG) would have developed. Complete disappearance of HNK-1 positive cells was evident already 10 hr after silastic implantation, before early differentiation sensory neurons could have reached their peripheral targets. In older embryos, DRG were absent at the level of implantation. In contrast, the development of ventral roots, sympathetic ganglia and adrenal gland was normal, and so was somitic differentiation into cartilage and muscle, while morphogenesis of the vertebrae was perturbed. To overcome the experimentally induced crest cell death, the silastic membranes were impregnated with a 3-day-old embryonic chick neural tube extract. Under these conditions, crest cells which were separated from the tube survived for a period of 30 hr after operation, compared to less than 10 hr in respective controls. The extract of another tissue, the liver, did not protract survival of DRG progenitor cells. Among the cells which survived with neural tube extract, some even succeeded in extending neurites; nevertheless, in absence of normal connections with the central nervous system (CNS) they finally died. Treatment of silastic implanted embryos with nerve growth factor (NGF) did not prevent the experimentally induced crest cell death. These results demonstrate that DRG develop from a population of neural crest cells which depends for its survival and probably for its differentiation upon a signal arising from the CNS, needed as early as the first hours after initiation of migration. Recovery experiments suggest that the subpopulation of crest cells which will develop along the sensory pathway probably depends for its survival and/or differentiation upon a factor contained in the neural tube, which is different from NGF.     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons

Studies were carried out in dissociated cell cultures on the nerve growth factor (NGF) requirement of chick embryo dorsal root ganglionic (DRG) neurons. Findings were: (i) The minimum level of 2.5 S NGF required to sustain the survival of maximal numbers of process-bearing cells derived from 8-day (E8) embryonic DRGs is 0.5 ng/ml (～2 × 10^?11M). (ii) Cultures derived from chick embryos of increasing ages (E8 to E18) showed a progressive increase in the proportion of process-bearing cells which survived in the absence of NGF. While few process-bearing cells survived in cultures of E8 ganglia in the absence of NGF, survival of neurons in cultures derived from E17 and E18 ganglia was not affected by the absence of the factor. Comparable results were obtained with cultures in which the number of non-neuronal cells was greatly reduced. (iii) Neurons derived from E8 ganglia lost their NGF requirement in culture at a conceptual age similar to that which they appear to do so in vivo. These results are discussed with respect to the role of NGF in development of sensory neurons.     

A quantitative study of the developmental expression of nerve growth factor (NGF) receptor in rats

The developmental expression of nerve growth factor (NGF) receptor was quantitated in either homogenates or plasma membrane-enriched preparations from whole rat embryos or from isolated tissues. The assay involved crosslinking 125I-NGF to receptors followed by immunoprecipitation with a monoclonal antibody to rat NGF receptor. In some cases, the pellet was resuspended and subjected to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiographic analysis. The NGF receptor was found in whole embryo homogenates as early as embryonic Day 10 (E10) (earliest age examined). The NGF receptor content in whole embryos per milligram protein increased about 3-fold from E11 to E18 and decreased slightly at E20. SDS-PAGE autoradiography showed that the molecular weights of 125I-NGF-bound receptors did not vary with age. The NGF receptor content in sciatic nerve homogenates decreased 23-fold from newborn to adulthood. The change of NGF receptor level in hindleg muscle had a profile similar to that seen in sciatic nerve. The NGF receptor content in superior cervical ganglion (SCG) or dorsal root ganglion (DRG) homogenate preparations was expressed in two ways. On a per milligram protein basis, in SCG, the receptor density was decreased slightly from E20 to adulthood; in DRG, it was relatively constant from E15 through postnatal Day 0 (PND-0) and then dropped 6.7-fold in adults. On a per ganglion basis, in SCG, it increased 4.4-fold from E20 to adult; in DRG, it increased 9-fold from E15 to PND-0 and then stayed constant through adulthood. In brain membrane preparations, the NGF receptor level decreased 11-fold from E15 to adulthood. In spinal cord membrane preparations, it decreased 7-fold from E18 to adulthood. Levels of receptor in cord were always greater than in brain. These data suggest that alterations in the NGF receptor density may have a role in changes in tissue responsiveness to NGF during development.     

Nerve growth factor-mediated induction of tyrosine hydroxylase in rat superior cervical ganglia in vitro.

Exposure of rat sympathetic ganglia to 3 microgram/ml of 2.5 S nerve growth factor (NGF) resulted in a 100% increase in tyrosine hydroxylase activity within 48 h. Pulselabeling of proteins with [3H]leucine, followed by immunoprecipitation with antibodies to tyrosine hydorxylase and isolation of the precipitated enzyme by gel electrophoresis, demonstrated that the increase in tyrosine hydroxylase activity was due to enhanced de novo synthesis. The incorporation of [3H]leucine into tyrosine hydroxylase was increased by 150% compared to a 17% increase in total protein synthesis, which was not statistically significant. The fact that the half-life of pulse-labeled tyrosine hydroxylase was the same for NGF-treated and control organ cultures of superior cervical ganglia excludes the possibility that enhanced tyrosine hydroxylase labeling by NGF is due to decreased degradation. We conclude that, without modulatory factors which play a role in vivo, NGF can enhance the synthesis of tyrosine hydroxylase in sympathetic ganglia in vitro, provided organ culture conditions which permit optimal survival of adrenergic neurons are selected.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

High K+-mediated survival of spinal sensory neurons depends on developmental stage

Elevated concentrations of K+ (35 mM) have previously been shown to support the survival of most embryonic chick sympathetic neurons in vitro (Wakade et al., Exp cell res 144 (1983) 377, [23]) and to be interchangeable with nerve growth factor (NGF) as a survival-promoting agent for these cells (Wakade & Thoenen, Neurosci lett 45 (1984) 71 [21]). In the present study, we show that dorsal root ganglion (DRG) neurons from embryonic day 6 do not survive in the presence of high K+, although both NGF and brain-derived neurotrophic factor (BDNF) each support the survival of more than 50% of the cells at this developmental stage. At E6, high K+ appears to have a cytotoxic effect on BDNF-dependent neurons, and there is also considerable inhibition of neurite outgrowth. At a later developmental stage (E12), high K+ supports the survival of about 40% of DRG cells. This subpopulation of neurons is distinct from that supported by NGF (as evidenced by the additivity of these two agents), but partially overlaps with that supported by BDNF (i.e., the two agents are less than additive). At E12, only approx. 20% of the cells can be supported by either NGF or BDNF, with the rest depending exclusively on one or the other of these factors. This is in contrast to the situation at E6, where there is considerable overlap between NGF- and BDNF-dependent populations.     

Rapid Activation of Tyrosine Hydroxylase in Response to Nerve Growth Factor

Abstract: Nerve growth factor protein (NGF) was found to rapidly promote the activation of tyrosine hydroxylase in cultured rat PC 12 pheochromocytoma cells. PC 12 cultures were exposed to NGF for periods of less than 1 h and the soluble contents of homogenates prepared from the cells were assayed for tyrosine hydroxylase activity. Under these conditions, the specific enzymatic activity was increased by 60 ± 10% (n = 13) in comparison with that in untreated sister cultures. The increase was half maximal by 2–5 min of exposure and at NGF concentrations of about 10 ng/ml (0.36 n M ). Antiserum against NGF blocked the effect. Tyrosine hydroxylase activity could also be rapidly increased by NGF in cultures of PC12 cells that had been treated with the factor for several weeks in order to produce a neuron-like phenotype. This was achieved by withdrawing NGF for about 4 h and then readding it for 30 min. The NGF-induced increase of tyrosine hydroxylase activity in PC12 cultures was not affected by inhibition of protein synthesis and therefore appeared to be due to activation of the enzyme. Kinetic experiments revealed that NGF brought about no change in the apparent K_m of the enzyme for tyrosine or for co-factor (6-methyltetrahydropteridine), but that it did significantly increase the apparent maximum specific activity of the enzyme. These observations suggest that NGF (perhaps released by target organs) could promote a rapid and local enhancement of noradrenergic transmission in the sympathetic nervous system.     

The thoracic sympathetic neurons of the chick: normal development and the effects of nerve growth factor

The generation and degeneration of sympathetic neurons in the third thoracic ganglion (segment 19) of the chick were studied between embryonic days (E) 7-18 using 3H-Thymidine autoradiography and routine cell counts. Cumulative radiolabelling experiments indicated that few sympathetic neurons were generated on E6-7. 10% of the sympathetic neurons were generated on E8 and a further 20% on E9. The final 70% of neurons completed the mitotic cycle between E10-12. Cell counts demonstrated that the neuronal population increased from 10,166 +/- 423 (mean +/- SEM) to 22,291 +/- 767 between E8-10 and remained stable up to E14. The population subsequently declined by 37%, to 14,157 +/- 831, by E18. Pyknotic neurons were found at all stages of development, but were most apparent between E7-15. The effects of Nerve Growth Factor (NGF) on the number of both surviving and pyknotic neurons in the ganglion were also examined. E9 embryos treated with NGF from E5-8 showed a 57% increase in the number of sympathetic neurons. This increase therefore occurred prior to the decline in neuronal number and was not accompanied by a decrease in the number of visibly pyknotic neurons. It is therefore possible that early NGF treatment increases the number of sympathetic neurons through a mechanism other than the attenuation of cell death.     

Characterization of a neuronal growth factor from mouse heart-cell-conditioned medium

A fraction of medium conditioned by embryonic mouse heart cells in culture promotes the growth of sympathetic and parasympathetic neurons in vitro. The factor stimulates neurite outgrowth, elevates specific activities of tyrosine hydroxylase and choline acetyltransferase in sympathetic ganglion explants, and enhances survival of dissociated sympathetic neurons in culture. The growth-promoting activity, which has a profound effect on survival of mouse sympathetic and parasympathetic neurons but little effect on mouse sensory neuron survival, is sensitive to trypsin and elevated temperature, suggesting association with a polypeptide or protein. Unlike nerve growth factor (NGF), the conditioned medium fraction is insensitive to anti-NGF antiserum, and fosters growth of mouse parasympathetic neurons. Consequently, the conditioned medium appears to contain a new nerve growth-promoting factor.     

Ionic behaviors and neuronal survival in developing ganglia. III. Studies with embryonic chick sympathetic neurons

We have shown in the past that (1) Nerve Growth Factor (NGF) controls the Na⁺,K⁺-pump in its ganglionic neuronal targets and (2) the NGF requirement for pump control is developmentally regulated in the chick embryo dorsal root ganglion. We report here that NGF is fully competent to insure the control of intracellular Na⁺ concentrations (as expression of pump control) in intact chick sympathetic ganglia and enriched suspensions of sympathetic neurons from embryonic day 8 (E8) through 13. At later stages (E13–E18), NGF becomes less and less required for that control as the neurons gain a self-sustained ionic pump competence. In monolayer cultures of enriched sympathetic neurons, an increasing neuronal survival in the absence of NGF occurs. These data demonstrate that the ability of developing sympathetic neurons to survive without NGF increases with the same temporal pattern as does their independence from NGF for ionic pump control, stressing the importance of ionic events for neuronal survival.     

Role of neurotrophin signalling in the differentiation of neurons from dorsal root ganglia and sympathetic ganglia

Manipulation of neurotrophin (NT) signalling by administration or depletion of NTs, by transgenic overexpression or by deletion of genes coding for NTs and their receptors has demonstrated the importance of NT signalling for the survival and differentiation of neurons in sympathetic and dorsal root ganglia (DRG). Combination with mutation of the proapoptotic Bax gene allows the separation of survival and differentiation effects. These studies together with cell culture analysis suggest that NT signalling directly regulates the differentiation of neuron subpopulations and their integration into neural networks. The high-affinity NT receptors trkA, trkB and trkC are restricted to subpopulations of mature neurons, whereas their expression at early developmental stages largely overlaps. trkC is expressed throughout sympathetic ganglia and DRG early after ganglion formation but becomes restricted to small neuron subpopulations during embryogenesis when trkA is turned on. The temporal relationship between trkA and trkC expression is conserved between sympathetic ganglia and DRG. In DRG, NGF signalling is required not only for survival, but also for the differentiation of nociceptors. Expression of neuropeptides calcitonin gene-related peptide and substance P, which specify peptidergic nociceptors, depends on nerve growth factor (NGF) signalling. ret expression indicative of non-peptidergic nociceptors is also promoted by the NGF-signalling pathway. Regulation of TRP channels by NGF signalling might specify the temperature sensitivity of afferent neurons embryonically. The manipulation of NGF levels “tunes” heat sensitivity in nociceptors at postnatal and adult stages. Brain-derived neurotrophic factor signalling is required for subpopulations of DRG neurons that are not fully characterized; it affects mechanical sensitivity in slowly adapting, low-threshold mechanoreceptors and might involve the regulation of DEG/ENaC ion channels. NT3 signalling is required for the generation and survival of various DRG neuron classes, in particular proprioceptors. Its importance for peripheral projections and central connectivity of proprioceptors demonstrates the significance of NT signalling for integrating responsive neurons in neural networks. The molecular targets of NT3 signalling in proprioceptor differentiation remain to be characterized. In sympathetic ganglia, NGF signalling regulates dendritic development and axonal projections. Its role in the specification of other neuronal properties is less well analysed. In vitro analysis suggests the involvement of NT signalling in the choice between the noradrenergic and cholinergic transmitter phenotype, in the expression of various classes of ion channels and for target connectivity. In vivo analysis is required to show the degree to which NT signalling regulates these sympathetic neuron properties in developing embryos and postnatally. U.E. is supported by the DFG (Er145-4) and the Gemeinnützige Hertie-Stiftung.     

Expression of recombinant human nerve growth factor in Escherichia coli.

Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons and may be of benefit in neurodegenerative diseases of humans. A method is described to obtain significant amounts of biologically active recombinant human NGF (rhNGF) in one step. RhNGF was expressed in E. coli and the majority of the protein accumulated in inclusion bodies. It was immunoprecipitated by a serum against mouse NGF. Solubilization of the inclusion bodies was done in 3M guanidine HCl and renaturation was effected by dilution and air oxidation in the presence of 6 microM CuSO4. Recoveries were 10-12 micrograms of rhNGF per ml of bacterial suspension. Its biological activity was tested in a bioassay system employing sympathetic chick embryo ganglia and was inhibited by the monoclonal antibody 27/21 against mouse NGF.