Death of developing septal cholinergic neurons following NGF withdrawal in vitro: protection by protein synthesis inhibition

Fetal septal neurons were grown in vitro under glass coverslips. This sandwich culture method significantly increased general neuronal survival, reduced glial proliferation, and permitted the removal of serum from the growth medium after 5 d in vitro. Thereafter, a simple, and completely defined, medium was used, and the effects of NGF, NGF withdrawal, and protein synthesis inhibition were examined on septal cholinergic neurons. NGF added to septal cultures at the time of plating resulted in a threefold increase in the number of cholinergic neurons seen at 14 d in vitro but had no effect on the survival of non-cholinergic cells. Cholinergic neurons identified by staining for AChE, ChAT, and p75NGFR could be maintained in serum-free, NGF-supplemented medium for over 40 d. When NGF was removed and NGF antibodies added to 14-d-old cultures, less than 30% of cholinergic neurons survived a further 4 d, but when NGF was similarly withdrawn from 35-d-old cultures, over 75% of cholinergic neurons survived. Reapplication of NGF after 3 but not after 12 or more hours of NGF withdrawal from 14-d-old cultures prevented the death of most cholinergic neurons. When NGF was withdrawn from 14-d-old cultures in the presence of the protein synthesis inhibitor cycloheximide, over 75% of the cholinergic neurons survived. These findings suggest that septal cholinergic neurons are dependent on NGF for survival only during a critical period of development and that growth factor-regulated developmental cell death may occur in CNS neurons by activation of programmed cell death requiring protein synthesis.     

Neurotrophin effects on survival and expression of cholinergic properties in cultured rat septal neurons under normal and stress conditions

These studies tested the hypothesis that survival-promoting effects of neurotrophins on basal forebrain cholinergic neurons are enhanced under stress. Septal neurons from embryonic day 14-15 rats exposed for 10-14 d to neurotrophin [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4 (NT-4), each at 100 ng/ml] showed a two- to threefold increase in choline acetyltransferase (ChAT) activity, with little evidence of synergistic interactions. Neurotrophins produced no significant increase in the survival of total or acetylcholinesterase (AChE)-positive neurons at moderate plating density (1200-1600 cells/mm2). However, with very low plating densities (2-28 cells/mm2) BDNF, NT-3, and NT-4 (but not NGF) increased total neuronal survival, and BDNF increased survival of AChE-positive neurons. NGF and BDNF enhanced ChAT activity and survival of cholinergic neurons after a 24 hr hypoglycemic stress, even when added 1 hr after stress onset. All four tested neurotrophins increased total neuronal survival after hypoglycemic stress. These results suggest that neurotrophins are important for preservation of central cholinergic function under stress conditions, with different neurotrophins protecting against different stresses. The stress-associated survival-promoting effects of neurotrophins were not limited to the cholinergic subpopulation.     

Pituitary adenylyl cyclase-activating polypeptide and nerve growth factor use the proteasome to rescue nerve growth factor-deprived sympathetic neurons cultured from chick embryos

Removal of nerve growth factor (NGF) from sympathetic neurons initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase-activating polypeptide (PACAP) prevents apoptosis in NGF-deprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prevented cell death for the next 24-48 h. Uptake of [3H]norepinephrine ([3H]NE) was used as an index of survival and decreased >50% in NGF-deprived cultures within 24 h. PACAP (1-100 nM) restored [3H]NE uptake to 92 +/- 8% of that of NGF-supported controls. Depolarization-induced [3H]NE release in neurons rescued by PACAP was the same as that in NGF-supported neurons. PACAP rescue was not mimicked by forskolin or 8-bromo-cyclic AMP and was not blocked by the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF-deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis-associated caspase CPP32 activity increased approximately fourfold during 6 h of NGF withdrawal (145 +/- 40 versus 38 +/- 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF-deprived, PACAP-rescued cultures (14 +/- 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF-deprived cultures reversed CPP32 activation, and this was blocked by lactacystin, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.     

Autocrine hepatocyte growth factor provides a local mechanism for promoting axonal growth

In this report, we describe a novel local mechanism necessary for optimal axonal growth that involves hepatocyte growth factor (HGF). Sympathetic neurons of the superior cervical ganglion coexpress bioactive HGF and its receptor, the Met tyrosine kinase, both in vivo and in vitro. Exogenous HGF selectively promotes the growth but not survival of cultured sympathetic neurons; the magnitude of this growth effect is similar to that observed with exogenous NGF. Conversely, HGF antibodies that inhibit endogenous HGF decrease sympathetic neuron growth but have no effect on survival. This autocrine HGF is required locally by sympathetic axons for optimal growth, as demonstrated using compartmented cultures. Thus, autocrine HGF provides a local, intrinsic mechanism for promoting neuronal growth without affecting survival, a role that may be essential during developmental axogenesis or after neuronal injury.     

Role of Leukotriene-degrading enzymes in pulmonary response to antigen infusion in sensitized guinea pigs in vivo

Although the expression of nerve growth factor (NGF) in the rat striatum is the highest at 2 postnatal weeks (P2w), the action of NGF at that age has not been studied in detail. We examined the effects of several neurotrophic factors, including NGF, on striatal cholinergic neurons cultured from P2w rats. We also examined the effects of a cyclic AMP (cAMP) analog and high K(+)-evoked depolarization. NGF specifically promoted the survival of choline acetyltransferase (ChAT)-positive neurons, and consequently increased the ChAT activity per well, whereas it did not induce the ChAT activity per cholinergic neuron. NGF-responsiveness was the highest in striatal cultures from P2w rats, but it was almost lost in cultures from P4w rats. Brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), and a cAMP analog had survival-promoting effects on striatal total neurons including cholinergic neurons. On the other hand, high K+ hardly promoted the survival of striatal cholinergic neurons in cultures from P2w rats, although it increased the viable number of total striatal neurons. High K+ did not increase the ChAT activity in any tested cultures from postnatal 3- to 28-day-old rats. These results demonstrated that NGF prevented the death of striatal cholinergic neurons in cultures from P2w rats, but not from P4w rats, and that high K+ could not rescue these deaths. We propose that cholinergic neurons in the striatum are programmed to die at P2w, and that this programmed cell death can be restored by neurotrophins, but not by depolarization.     

Sympathetic neurons expressing cholinergic properties are poised to allocate choline symmetrically between acetylcholine and the phosphatidylcholine-generating pathway in growing neurites

We have examined the question of how regenerating sympathetic neurons that are concomitantly induced to become cholinergic regulate choline allocation between ACh and the phospholipid synthetic pathway. The allocation of choline into ACh increased parabolically with time in culture, and by 3 weeks, cultures with neurites of approximately 6 mm length were incorporating over 85% of the choline locally in the neurites into four major metabolites: ACh, phosphorylcholine, cytidine diphosphocholine, and phosphatidylcholine. The near-equivalent distribution of labeled choline between intracellular choline, ACh, and phosphorylcholine was independent of time (5 min to 6 hr) and choline concentration (0.125-30 microM), phosphatidylcholine being the sole metabolite whose level in the neurites increased steadily with incubation time. Relative choline distribution into ACh and phosphorylcholine was unaltered even after a brief depolarizing prepulse, which caused a two- to fourfold enhancement in the total choline incorporated. These observations, allied with the similar half-saturation constants and Vmax values of CAT and choline kinase for intracellular choline, suggest that growing sympathetic neurons are poised to allocate choline symmetrically between the synthesis of ACh and phosphatidylcholine in the neurites. When, however, the supply of choline was limited either by replacement of Na+ in the medium with N-methyl-D-glucamine, or by vesamicol, a 90-97% reduction in intracellular choline caused a similar decline in ACh levels but synthesis of metabolites of the phosphatidylcholine pathway was maintained unperturbed, as if no drug was present. We suggest that this can be accounted for by a 10-fold increase in choline kinase activity. Thus, growing sympathetic neurons that express cholinergic properties not only maintain their chief cellular phosphatidylcholine-synthesizing activity concomitantly with ACh synthesis in the neurites, but may also preserve phosphatidylcholine synthesis more effectively than ACh synthesis when the supply of choline is perturbed. Relinquishing ACh synthesis during growth may be one way of conserving and encouraging neurite regeneration.     

Effects of nerve growth factor on glutathione peroxidase and catalase in PC12 cells

Nerve growth factor (NGF) is a member of the neurotrophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione peroxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC12 cells in a time- and dose-dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.     

Cell death of neonatal rat sensory neurons is prevented by culture at clonal density

We studied the trophic requirements of neonatal rat sensory neurons in single neuron and standard culture (9400 neurons/cm2). In agreement with previous studies, we found that survival of sensory neurons in standard culture is strictly dependent on nerve growth factor (NGF). Contrary to this, however, no difference between NGF-treated and untreated cultures with respect to survival and neurite formation was evident when neurons were plated as single cells. Approximately 70% of singly-seeded sensory neurons survived for 2 days independent of supply with NGF. Survival of large, mid-sized, and small sensory neurons in single neuron culture demonstrated that this phenomenon is not confined to a neuronal subpopulation. We speculate that survival in single neuron culture is due to the absence or inactivation of yet unidentified cell death-inducing factors that in standard culture are overridden or suppressed by NGF.     

Regulation of galanin in rat sympathetic neurons in vitro

The neuropeptide galanin is induced in sensory and autonomic neurons after peripheral nerve lesion. Leukemia inhibitory factor (LIF) has been suggested to be involved in the up-regulation of galanin. A direct effect of LIF on galanin content in pure sympathetic neuron cultures dissociated from newborn rat superior cervical ganglia was investigated by radioimmunoassay and immunohistochemistry. Galanin increases in sympathetic neurons during a 12 day culture period in the presence of NGF (10 ng/ml). Five days after addition of LIF (10 ng/ml) a 7-fold elevation is observed when compared to control cultures. Furthermore, galanin increases significantly in the presence of non-neuronal cells and in response to potassium-induced depolarization. The proportion of galanin-immunoreactive neurons in mixed cultures is similar to that found in adult rat superior cervical ganglia after transection of the major postganglionic branches. The results corroborate the hypothesis that LIF, presumably released from ganglionic satellite cells, induces galanin in a subpopulation of sympathetic neurons in vivo and in vitro.     

NGF binding to p75 enhances the sensitivity of sensory and sympathetic neurons to NGF at different stages of development

To clarify the role of the common neurotrophin receptor p75 in modulating the survival response of sensory and sympathetic neurons to NGF at different stages of development, we compared the actions of wild-type NGF with a mutated NGF protein that binds normally to TrkA, the NGF receptor tyrosine kinase, but has greatly reduced binding to p75. At saturating concentrations, the NGF mutant promoted the survival of similar numbers of trigeminal sensory and sympathetic neurons as NGF. At subsaturating concentrations, the NGF mutant was less effective than wild-type NGF in promoting the survival of embryonic sensory neurons and postnatal sympathetic neurons but was equally effective as wild-type NGF in promoting the survival of embryonic sympathetic neurons. Whereas the levels of trkA and p75 were similar in embryonic sensory neurons and postnatal sympathetic neurons, the level of p75 was significantly lower than that of trkA in embryonic sympathetic neurons. These results indicate that binding of NGF to p75 enhances the sensitivity of NGF-dependent neurons to NGF at stages in their development when the levels of p75 and TrkA are similar.     

Rapid suppression of free radical formation by nerve growth factor involves the mitogen-activated protein kinase pathway

Neurotrophins such as nerve growth factor (NGF) regulate neuronal survival during development and are neuroprotective in certain models of injury to both the peripheral and the central nervous system. Although many effects of neurotrophins involve long-term changes in gene expression, several recent reports have focused on rapid effects of neurotrophins that do not involve synthesis of new gene products. Because enhanced formation of reactive oxygen species (ROS) represents one consequence of many insults that produce neuronal death, we hypothesized that neurotrophins might influence neuronal function and survival through acute alterations in the production of ROS. Using an oxidation-sensitive compound, dihydrorhodamine, we measured ROS formation in a central nervous system-derived neuronal cell line (GT1-1 trk) and in superior cervical ganglion neurons, both of which express the transmembrane NGF receptor tyrosine kinase, trkA. There was enhanced production of ROS in both cell types in the absence of NGF that was rapidly inhibited by application of NGF; complete inhibition of ROS generation in GT1-1 trk cells occurred within 10 min. NGF suppression of ROS formation was prevented by PD 098059, a specific inhibitor of MEK (mitogen/extracellular receptor kinase, which phosphorylates mitogen-activated protein kinase). The observation that NGF acutely blocks ROS formation in neurons through activation of the mitogen-activated protein kinase pathway suggests a novel mechanism for rapid neurotrophin signaling, and has implications for understanding neuroprotective and other effects of neurotrophins.     

Constitutive secretion of exogenous neurotransmitter by nonneuronal cells: implications for neuronal secretion

Fibroblasts in cell culture were loaded with exogenous neurotransmitter acetylcholine (ACh). ACh secretion from loaded cells was detected by whole-cell patch clamp recordings from Xenopus myocytes manipulated into contact with ACh-loaded cells. Two different approaches were used for ACh loading. In the first approach, fibroblasts were incubated in the culture medium containing ACh. Recordings from myocytes revealed fast inward currents that resemble miniature endplate currents found at neuromuscular synapses. The currents observed in recordings from myocytes were due to exocytosis of ACh-containing vesicles. Although exogenous ACh penetrated through the plasma membrane of fibroblasts during incubation and was present in the cytoplasm at detectable levels, cytoplasmic ACh did not contribute to the quantal ACh secretion. In the second approach, exogenous ACh was loaded into the cytoplasm of fibroblasts by microinjection. Under these experimental conditions, fibroblasts also exhibited spontaneous quantal ACh secretion. Analysis of the exocytotic events in fibroblasts following two different protocols of ACh loading revealed that the vesicular compartments responsible for uptake of exogenous ACh are associated with the endocytic recycling pathway. Extrapolation of our results to neuronal cells suggest that in cholinergic neurons, in addition to genuine synaptic vesicles, ACh can be secreted by the vesicles participating in endosomal membrane recycling.     

The developmental expression of choline acetyltransferase (ChAT) and the neuropeptide VIP in chick sympathetic neurons: evidence for different regulatory events in cholinergic differentiation

Cholinergic properties in chick sympathetic neurons are detectable early during development of paravertebral ganglia and mature after target contact. The cholinergic marker choline acetyltransferase (ChAT) is first detectable at embryonic day 6 and its expression partly overlaps with that of the noradrenergic marker tyrosine hydroxylase (TH). At late embryonic stages, when sympathetic neurons have established target contact, ganglia consist of two major neuronal populations, TH-positive noradrenergic neurons and cholinergic neurons that at this stage express vasoactive intestinal peptide (VIP) in addition to ChAT. The maturation of sympathetic neurons is paralleled by changes in their response to the neurokine ciliary neurotrophic factor (CNTF). These findings suggest that expression of neurotransmitter properties is controlled differentially before and during target innervation.     

Production of sweat gland cholinergic differentiation factor depends on innervation

Sympathetic neurons innervating sweat glands undergo a target-directed developmental switch in neurotransmitter properties. Using cultured sympathetic neurons as a bioassay for cholinergic differentiation factors, we and others found that extracts containing soluble proteins from developing and adult footpads caused the same changes in transmitter properties in sympathetic neurons in vitro that the target does in vivo. In the present studies, using footpads from Tabby mutant mice that lack sweat glands, we found that the presence of sweat glands is correlated with the presence of cholinergic differentiation activity in footpad extracts. We examined the conditions necessary for secretion of differentiation activity from primary cultures of sweat gland cells. Surprisingly, sweat gland cells cultured alone do not produce or secrete cholinergic differentiation activity. When grown in the presence of sympathetic neurons, however, gland cells induce cholinergic function, increase vasoactive intestinal peptide content, and reduce catecholamine production in the neurons. Medium conditioned by sweat gland/neuron cocultures has a similar effect on the transmitter properties of cultured sympathetic neurons, indicating that the target influence on phenotype is mediated by a secreted factor(s). The innervation-dependence of cholinergic differentiation factor production provides evidence that reciprocal interactions between neurons and sweat glands are necessary for acquisition of the mature transmitter phenotype.     

Bone morphogenetic protein-2 and retinoic acid induce neurotrophin-3 responsiveness in developing rat sympathetic neurons

Expression of the receptor tyrosine kinase, Trk, determines the specificity of neurotrophin responsiveness of different neuronal populations during development. Recently it has become apparent that sympathetic neurons of rat superior cervical ganglia (SCG) acquire sensitivity to neurotrophin-3 (NT3) before they become dependent on the target-derived nerve growth factor (NGF) for their survival by sequential induction of TrkC and TrkA. The mechanism controlling the expression of TrkC as well as the source of NT3 at their initial developmental stage has, however, not been clarified. Here we show that the treatment of the perinatal rat SCG neurons which express high levels of trkA mRNA with bone morphogenetic protein-2 (BMP2) induced the expression of trkC mRNA. Induction of the functional TrkC receptor by BMP2 was confirmed by the enhancement of the survival response of these neurons to NT3. Treatment of SCG neurons with retinoic acid (RA) promoted the effect of BMP2 on the induction of trkC mRNA levels. BMP2 treatment, on the other hand, promoted the effect of RA on the suppressions of trkA mRNA levels and the NGF-dependent survival of the SCG neurons. Furthermore, BMP2/RA treatment induced the endogenous expression of NT3. These results indicate that specific environmental signals can regulate neurotrophin responsiveness of developing sympathetic neurons by differential alteration of the trk and neurotrophin expressions.     

Age-related effects of nerve growth factor on the morphology of embryonic sensory neurons in vitro

Studies of neonatal and adult mammals have shown that neuronal morphology is regulated in part by the availability of target-derived neurotrophic factor. To test whether the same is true for embryonic neurons, which are dependent on target-derived neurotrophic factors for survival, we grew neural crest-derived sensory neurons from the trigeminal ganglion of avian embryos of different ages in vitro in different concentrations of nerve growth factor (NGF) and measured the number of branch points and total length of the resulting arborizations. Although the size and complexity of arborizations increased with embryonic age up to embryonic day (E)14, neuronal morphology for embryos younger than E14 was unaffected by the concentration of NGF in the culture medium. However, beginning at E14, the stage at which trigeminal neurons start to lose their absolute requirement for NGF for survival, the neurons had significantly more branch points and larger arborizations in higher concentrations of NGF. Thus, it appears that the extent of neurite outgrowth in young embryos is independent of neurotrophic factor concentration; each neuron that receives enough neurotrophic factor to survive elaborates approximately the same size arbor. As trigeminal neurons mature and become less dependent on neurotrophic factor for survival, they acquire the ability to respond to neurotrophic factor with increased neurite growth and branching, as in neonates and adults.     

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cgamma1 (PLCgamma1). We used recombinant glutathione-S-transferase (GST)-PLCgamma1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCgamma1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology-2 (SH2) domain of PLCgamma1, but there was enhanced binding with tandemly expressed amino- and carboxy-terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen-activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.     

p53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors

Naturally occurring sympathetic neuron death is the result of two apoptotic signaling events: one normally suppressed by NGF/TrkA survival signals, and a second activated by the p75 neurotrophin receptor. Here we demonstrate that the p53 tumor suppressor protein, likely as induced by the MEKK-JNK pathway, is an essential component of both of these apoptotic signaling cascades. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75NTR activation. NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax. Functional ablation of p53 using the adenovirus E1B55K protein inhibits neuronal apoptosis as induced by either NGF withdrawal or p75 activation. Direct stimulation of the MEKK-JNK pathway using activated MEKK1 has similar effects; p53 and Bax are increased and the subsequent neuronal apoptosis can be rescued by E1B55K. Expression of p53 in sympathetic neurons indicates that p53 functions downstream of JNK and upstream of Bax. Finally, when p53 levels are reduced or absent in p53+/- or p53-/- mice, naturally occurring sympathetic neuron death is inhibited. Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.