Increased expression of T alpha 1 alpha-tubulin mRNA during collateral and NGF-induced sprouting of sympathetic neurons

We have examined expression of T alpha 1 alpha-tubulin mRNA in the rat superior cervical ganglion (SCG) to determine whether changes in gene expression accompany neuronal sprouting and to investigate factors that regulate growth-associated genes in intact neurons. Northern blot analysis demonstrates that levels of T alpha 1 alpha-tubulin mRNA increase in the uninjured SCG following transection of contralateral neurons that project to bilaterally innervated, but not unilaterally innervated target organs. The observed increase in uninjured neurons is associated with collateral sprouting, as measured by increased tyrosine hydroxylase immunoreactivity within the pineal gland. These data suggest that target-derived factors may regulate T alpha 1 mRNA in sprouting neurons. Consistent with this hypothesis, systemic NGF treatment of neonatal animals over a developmental interval when T alpha 1 alpha-tubulin mRNA normally decreases led to a 5- to 10-fold increase in T alpha 1 mRNA levels in developing sympathetic neurons. In addition, deafferentation of the SCG, which promotes neuronal sprouting in the ganglion, increases T alpha 1 mRNA in ganglia on the ipsilateral and contralateral sides. Together, these data demonstrate that T alpha 1 alpha-tubulin mRNA elevates as a function of neuronal sprouting, and that T alpha 1 mRNA expression in intact neurons can be regulated by extrinsic cues, including NGF and changes in connectivity.     

Isotypes of alpha-tubulin are differentially regulated during neuronal maturation

The mRNAs for two isotypes of alpha-tubulin, termed T alpha 1 and T26, are known to be expressed in the rat nervous system. We have compared the expression of these two alpha-tubulin mRNAs during neural development, using RNA blotting and in situ hybridization techniques with probes directed against unique sequences of each mRNA. T alpha 1 mRNA is highly enriched in the embryonic nervous system but is markedly less abundant in the adult brain; T26 mRNA is expressed in many embryonic tissues with little change in abundance during development. Within the nervous system, T alpha 1 mRNA is enriched in regions with neurons actively undergoing neurite extension, such as the cortical plate, whereas T26 mRNA is relatively homogeneous in distribution, with some enrichment in proliferative zones. Expression of T alpha 1 mRNA is also increased in PC12 cells induced to differentiate and extend neurite processes by nerve growth factor. Taken together, the data indicate that T alpha 1-tubulin mRNA is expressed at high levels during the extension of neuronal processes. The abundant expression of T alpha 1-tubulin mRNA may therefore reflect either a means to increase the available pool of alpha-tubulin or a specific requirement for the T alpha 1 isotype for neurite extension.     

Glial-cell-line-derived neurotrophic factor induces nerve fibre formation in primary cultures of adrenal chromaffin cells

Neurotrophic factors, such as nerve growth factor (NGF), have been shown to promote the differentiation of neural crest neuroblasts into sympathetic neurons, whereas glucocorticoids promote the endocrine phenotype of adrenal medullary chromaffin cells. This pluripotency is preserved to some extent in adult chromaffin cells, with NGF and other neurotrophic factors influencing the differentiation of these cells. In this study, the effects of glial cell line-derived neurotrophic factor (GDNF) on explanted chromaffin tissue have been investigated. The localization of mRNAs corresponding to the two components of the GDNF receptor, GDNF family receptor alpha 1 (GFRalpha1) and Ret, were demonstrated in adult adrenal medullary ganglion cells. GFRalpha1 mRNA was expressed in explanted chromaffin tissue at levels dependent on the presence of serum in the medium but decreased on the addition of blocking antibodies against transforming growth factor beta (TGFbeta). However, TGFbeta1 (1 ng/ml) did not upregulate GFRalpha1 mRNA expression when added to serum-free medium. GDNF induced neurite formation from chromaffin cells, as measured by the ratio of neurite-bearing versus total number of chromaffin cells in primary cultures of adult adrenal medulla. The most potent dose inducing neurites from chromaffin cells was 100 ng/ml GDNF. However, this dose was not as efficient as that seen when chromaffin cells were stimulated with NGF (100 ng/ml). Thus, adrenal medullary cells express mRNAs for the GDNF receptor components Ret and GFRalpha1, increase their expression upon being cultured in serum-containing medium and respond to GDNF treatment with an increase in the number of cells that develop nerve processes.     

Distinct spatial localization of specific mRNAs in cultured sympathetic neurons

We examined the subcellular distribution of specific mRNAs in cultured sympathetic neurons. Under appropriate conditions, sympathetic neurons extend both axons and dendrites that are distinguishable by light microscopic and immunocytochemical criteria. In situ hybridization revealed a differential localization of mRNA within dendrites. mRNA encoding MAP2 was abundant in cell bodies and distributed nonhomogeneously throughout the dendritic compartment, but was not detected in axons. In contrast, mRNAs encoding GAP-43 and alpha-tubulin were restricted to the cell body and largely excluded from dendrites as well as axons. Detergent extraction revealed that most dendrite-associated mRNA encoding MAP2 was associated with the Triton X-100 insoluble fraction of the cell. The subset of mRNAs present in the dendritic compartment may encode proteins involved in the morphogenesis and remodeling of dendrites.     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons: I. Sympathetic neurons

Quantitative studies on the nerve growth factor (NGF) requirement of chick embryo sympathetic neurons in dissociated cell culture revealed the following. (i) The minimum concentration of 2.5 S NGF required for survival of maximal numbers of neurons is about 0.5 ng/ml (～2 × 10^?11M). In culture, this concentration of NGF appears not to be stable for more than 24 hr. Long-term neuronal maintenance with medium changes twice weekly requires a minimum of 5 ng/ml of NGF. (ii) At 24 hr after plating in medium containing 10% fetal bovine serum, neuronal survival is less than optimal at NGF concentrations above 5 ng/ml; in medium with 5% horse serum, survival is constant with up to 5000 ng/ml of NGF. (iii) Survival of neurons after 1 week in culture was less than optimal at NGF concentrations greater than 50 ng/ml, even in medium containing horse serum. (iv) No correlation was observed between the level of NGF (0.5–500 ng/ml) and the estimated neuronal somatic volumes up to 1 month in vitro. (v) Withdrawal of NGF, even after 4 weeks of culture, resulted in degeneration of nerve cell bodies and processes.     

Effect of anti-NGF on ovarian expression of alpha1- and beta2-adrenoceptors, TrkA, p75NTR, and tyrosine hydroxylase in rats with steroid-induced polycystic ovaries

Estradiol valerate (EV)-induced polycystic ovaries (PCO) in rats are associated with higher ovarian release and content of norepinephrine, decreased beta2-adrenoceptors (ARs), and dysregulated expression of alpha1-AR subtypes, all preceded by an increase in the production of ovarian NGF. The aim of this study was to further elucidate the role of NGF in the ovaries by blocking the action of NGF during development of EV-induced PCO in rats. Control and EV-injected rats were treated with intraperitoneal injections of IgG (control and PCO groups) or with anti-NGF antibodies (anti-NGF and PCO anti-NGF groups) every third day for 5 wk starting from the day of PCO induction. Rat weight, estrous cyclicity, ovarian morphology, ovarian mRNA, and protein expression of alpha1-AR subtypes, beta2-AR, the NGF receptor tyrosine kinase A (TrkA), p75 neurotrophin receptor (p75NTR), and tyrosine hydroxylase (TH) were analyzed. Ovaries in both PCO and PCO anti-NGF groups decreased in size as well as in number and size of corpora lutea. mRNA expression of alpha1a-AR and TrkA in the ovaries was lower, whereas expression of alpha1b- and alpha1d-AR and TH was higher, in the PCO group than in controls. Protein quantities of alpha1-ARs, TrkA, p75NTR, and TH were higher in the PCO group compared with controls, whereas the protein content of beta2-AR was lower. Anti-NGF treatment in the PCO group restored all changes in mRNA and protein content, except that of alpha1b-AR and TrkA mRNAs, to control levels. The results indicate that the NGF/NGF receptor system plays a role in the pathogenesis of EV-induced PCO in rats.     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. III. Effect on acetylcholine production

The effect of nerve growth factor (NGF) on the development of cholinergic sympathetic neurons was studied in cultures grown either on monolayers of dissociated rat heart cells or in medium conditioned by them. In the presence of rat heart cells the absolute requirement of neurons for exogenous NGF was partially spared. The ability of heart cells to support neuronal survival was due at least in part to production of a diffusable NGF-like substance into the medium. Although some neurons survived on the heart cell monolayer without added NGF, increased levels of exogenous NGF increased neuronal survival until saturation was achieved at 0.5 microgram/ml 7S NGF. The ability of neurons to produce acetylcholine (ACh) from choline was also dependent on the level of exogenous NGF. In mixed neuron-heart cell cultures, NGF increased both ACh and catecholamine (CA) production per neuron to the same extent; saturation occurred at 1 microgram/ml 7S NGF. As cholinergic neurons developed in culture, they became less dependent on NGF for survival and ACh production, but even in older cultures approximately 40% of the neurons died when NGF was withdrawn. Thus, NGF is as necessary for survival, growth, and differentiation of sympathetic neurons when the neurons express cholinergic functions as when the neurons express adrenergic functions (4, 5).     

Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells.

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.     

Sympathetic neurons synthesize and secrete pro‐nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF‐immunoreactive proteins synthesized by cultured NGF‐dependent and ‐independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro‐NGF protein. These findings suggest that a potential NGF‐sympathetic neuron autocrine loop may exist in this prototypic target‐dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival. © 2003 Wiley Periodicals, Inc. J Neurobiol 38–53, 2003     

SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.     

Epidermal growth factor induces tyrosine hydroxylase in a clonal pheochromocytoma cell line, PC-G2

We have previously described the isolation of a clonal cell line (PC-G2) in which the level of tyrosine hydroxylase (TH), the rate-limiting step in the synthesis of the catecholamine neurotransmitters, is induced by nerve growth factor (NGF). We now report that epidermal growth factor (EGF) also induces TH in the PC-G2 cell line. Although EGF has been shown to be mitogenic for many cultured cells, no neuronal function has been previously reported for this protein. The TH response to EGF is elicited in a dose-dependent fashion at concentrations as low as 0.1 ng/ml and is maximal at 10 ng/ml EGF. The maximal response is observed after 3--4 d of exposure to 10 ng/ml EGF. The induction by NGF and EGF is inhibited by their respective antisera. Dexamethasone, a synthetic glucocorticoid which we have previously shown modulates the response of PC-G2 cells to NGF, also modulates the TH induction elicited by EGF.     

神经生长因子分化后的PC12 细胞对乙酰胆碱的敏感性及其特性

目的和方法：采用全细胞膜片钳技术观察神经生长因子（NGF）分化后的PC12细胞对乙酰胆碱（ACh)的敏感性,并对ACh诱发电流（IACh)的特性进行分析。结果：NGF处理后的PC12乐仅形态上向交感神经元分化,而且具有电学兴奋性,它对ACh敏感性比未分化前显著提高。药理学鉴定表明PC12上的IACh是由烟碱受体（nAChR)引起的,具有明显的失敏特性。宏观IACh呈内向整流和浓度依赖性。结论：PC12细胞培养方便,同源性好,加入NGF后向交感神经元分化,且其具有神经元烟碱受体,可以作为交感神经元烟碱受体研究的很好的模型系统。     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. I. Survival, growth, and differentiation of catecholamine production

To study the effect of nerve growth factor (NGF) on neuronal survival, growth, and differentiation, cultures of dissociated neonatal rat sympathetic neurons virtually free of other cell types were maintained for 3-4 wk. In the absence of NGF, the neurons did not survive for more than a day. Increased levels of NGF increased neuronal survival and growth (total protein and total lipid phosphate); saturation occurred at 0.5 microgram/ml 7S NGF. Neuronal differentiation examined by measuring catecholamine (CA) production from tyrosine also depended on the level of NGF in the culture medium. As the NGF concentration was raised, CA production per neuron, per nanogram protein, or per picomole lipid phosphate increased until saturation was achieved between 1 and 5 microgram/ml 7S NGF. Thus, NGF induces neuronal survival, growth, and differentiation of CA production in a dose-dependent fashion. Neuronal growth and differentiation were quantitatively compared in the presence of the high and low molecular weight forms of NGF; no significant functional differences were found.     

Sympathetic neurons synthesize and secrete pro-nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF-immunoreactive proteins synthesized by cultured NGF-dependent and -independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro-NGF protein. These findings suggest that a potential NGF-sympathetic neuron autocrine loop may exist in this prototypic target-dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival.     

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.