CEP-1347 promotes survival of NGF responsive neurones in primary DRG explants

CEP-1347 inhibits the signalling pathway of c-jun-N-terminal kinase, and is neuroprotective in vivo and in vitro. Embryonic chick dorsal root ganglion neurones are dependent on NGF for survival and neurite outgrowth; NGF withdrawal results in apoptotic cell death. We examined the neuroprotective and neurite outgrowth promoting activity of CEP-1347 in dissociated DRG neurones and in primary DRG explants. CEP-1347 was as effective as NGF in promoting survival of dissociated DRG neurones, but caused only limited neurite outgrowth from DRG explants. When NGF was subsequently added to CEP-1347 treated explants, the outgrowth increased to a similar level to explants which had received NGF throughout. CEP-1347 may be a useful tool to maintain viable DRG explants to allow evaluation of neurite outgrowth promoting compounds and dissection of survival and neurite outgrowth signalling pathways.     

Effect of nerve growth factor on the elongation of neurites from axotomized rat embryonic septal-basal forebrain neurons: an in vitro analysis

The administration of nerve growth factor (NGF) into the brain of a fornix-fimbria lesioned rat can rescue many cholinergic, septal-basal forebrain (SBF) neurons from imminent cell death. Unfortunately, it is unclear if NGF can stimulate regenerative growth from axotomized, SBF neurons. In the present study, we used an in vitro model system to determine if NGF could affect neurite outgrowth from nonaxotomized and/or axotomized, embryonic SBF neurons. Axotomized neurons were obtained by severing the neuritic fields surrounding embryonic day (E) 15 SBF explants maintained in primary culture. Acetylcholinesterase (AChE) histochemistry was used to assess the effects of NGF on cholinergic neurites. We report that 1) neurite outgrowth on type I collagen from E15 SBF neurons in primary culture (nonaxotomized neurons) was not affected by NGF. 2) NGF enhanced the outgrowth (regeneration) of axotomized, SBF neurons on a collagen substratum; however, neurons had to be treated with NGF both before and after axotomy to stimulate regeneration effectively. Application of NGF either before or after axotomy did not enhance regenerative neurite outgrowth. 3) SBF neurons had to be axotomized for NGF to facilitate neurite outgrowth. This is supported by the observation that SBF explants, initially maintained in NGF-supplemented medium in suspension culture, did not demonstrate enhanced neurite outgrowth in the presence of NGF when plated onto a substratum. 4) The regenerative growth of AChE-negative, as well as AChE-positive, neurites was facilitated by NGF treatment. In addition to data concerning neurite outgrowth, we also found that the NGF receptor, as recognized by the antibody 192-IgG, expands its distribution as time in culture progresses; i.e., staining, originally confined to cell bodies and proximal processes within the explant, later included neurites that emanated from the explant. Thus, our results demonstrate that NGF can stimulate regenerative growth from axotomized, but not nonaxotomized, embryonic SBF neurons. We hypothesize that, given the appropriate substratum for axon elongation in vivo, NGF can stimulate the regeneration of SBF neurons in the injured adult brain.     

Influence of nerve growth factor on developing dorso-medial and ventro-lateral neurons of chick and mouse trigeminal ganglia

Trigeminal ganglia have been removed from five, six, seven and eight day chick embryos and explants of the dorso-medial (DM) and ventro-lateral (VL) parts of the maxillomandibular lobe were grown in tissue culture. Quantitative methods were used to assess the influence of nerve growth factor (NGF) on fiber outgrowth from these explants. At all ages outgrowth from DM explants was significantly greater than from VL explants, the difference being most pronounced between the extreme DM and VL poles of the maxillomandibular lobe. These observations are interpreted as indicating the existence of two distinct populations of neurons in terms of their response to NGF rather than the consequence of the asynchronous differentiation and maturation of the VL and DM neurons. A similar study of 10, 11 and 12 day embryonic mouse trigeminal ganglia revealed no significant difference in neurite outgrowth between DM and VL regions grown in the presence or absence of NGF.     

Involvement of dihydropyridine-sensitive calcium channels in nerve growth factor-dependent neurite outgrowth by sympathetic neurons

We have used a number of pharmacological manipulations of calcium influx to alter the nerve growth factor (NGF)-elicited neurite outgrowth response of SCG neurons. Our results indicate that influx of extracellular calcium is critical to sympathetic SCG neurite outgrowth. Effective blockade of this process was produced by the inorganic calcium channel blockers Cd2+ (with an IC50 of 48 microM), Co2+ (129 microM), and Ni2+ (180 microM). More specifically, there is a significant contribution from dihydropyridine-sensitive L-type calcium channels to NGF-activated neurite outgrowth, as evidenced by the significant inhibition of neurite outgrowth by diltiazem (IC50 of 17 microM) and nifedipine (3 microM). Further, increases in calcium influx can elicit an enhanced neurite outgrowth response, as shown by the calcium channel agonist Bay K 8644 which potentiated neurite outgrowth by up to 40%.     

Secretion of nerve growth factor from septum stimulates neurite outgrowth and release of the amyloid protein precursor of Alzheimer's disease from hippocampal explants

Alzheimer's disease (AD) is characterized by the deposition of amyloid in the extracellular and intracellular compartments of the cerebral cortex. The extracellular amyloid consists of a protein (βA4) which is derived from a larger precursor, the amyloid protein precursor (APP). Several studies have implicated APP in the regulation of neurite outgrowth during development, although the precise function of APP remains unknown. To examine the role of APP in the regulation of neutrite outgrowth from hippocampal neurons, an explant culture system was developed. Explants of E18 mouse hippocampus were found to extend neurites when co-cultured with explants of E18 mouse septum. This finding demonstrated that the septum can release a neurite outgrowth-promoting factor (NOPF). As nerve growth factor (NGF) was also able to stimulate neurite outgrowth from the hippocampal explants, this suggested that the NOPF might be NGF. Immunoprecipitation of NGF from septal conditioned medium using a specific monoclonal antibody (27/21) completely blocked the neurite outgrowth-promoting effect, supporting this conclusion. Concomitant with its ability to stimulate neurite outgrowth, NGF stimulated the release of APP from the hippocampal explants. As previous studies have suggested that the binding of APP to heparan sulfate proteoglycans (HSPGs) in the extracellular matrix might be an important step in the regulation of neurite outgrowth by NGF, we examined the effect of APP on neurite outgrowth from dissociated hippocampal cells cultured on various protein substrates. When cells were cultured on a substrate of APP and HSPG, neurite outgrowth was markedly stimulated. No stimulation of neurite outgrowth was seen when neurons were cultured on substrates of either APP or HSPG alone. The results suggest that secreted forms of APP may be involved in stimulating neurite outgrowth from hippocampal neurons and that interactions between APP and HSPG may be important for a neurite outgrowth-promoting function. © 1994 Wiley-Liss, Inc.     

Retinal ganglion cell response to axotomy and nerve growth factor antiserum treatment in the regenerating visual system of the goldfish (Carassius auratus): An in vivo and in vitro analysis

In vitro nerve growth factor (NGF) antiserum (anti-NGF) treatment was found to significantly depress retinal ganglion cell neurite outgrowth in goldfish explant culture. Goldfish retinas, conditioned by a 14-day prior optic nerve crush, demonstrated a significant dose response inhibition of neurite outgrowth if incubated with various concentrations of the antiserum (i.e. concentrations from full strength to 1:100) before explanation for tissue culture. NGF added to the incubation medium containing antiserum partially eliminated the inhibition of neurite outgrowth during the first 4 days of explant culture. Antiserum treatment at the higher concentrations (i.e. full strength and 1:1.5 dilution) caused a cessation of nerve growth from explants between culture days 3 and 4. However, controls at this time still exhibited vigorous neurite outgrowth.In vivo treatment with anti-NGF administered intraocularly at 7 days after optic nerve crush (i.e. 7 DPA) was found to significantly reduce the size and complexity of retinal ganglion cell nucleoli when analyzed morphometrically at 14 DPA. No other cell parameters measured (i.e. cell size, nuclear size, cell/nuclear ratios and mitochondrial, Golgi and RER densities) were found to be affected by the single antiserum treatment.     

Serum- and substratum-dependent modulation of neuritic growth

Explants of embryonic day 8 (E8) chicken dorsal root ganglia (DRG) have been cultured with medium containing serum or the serum-free supplement N1 on one of three substrata: collagen, polyornithine (PORN), or PORN exposed to a polyornithine-binding neurite-promoting factor (PNPF-PORN). Replicate cultures were maintained with or without nerve growth factor (NGF). NGF elicited its classical neuritic outgrowth on all three substrata in serum-containing or serum-free medium. In the absence of NGF, however, a gradation of increasing neurite growth was seen with: PNPF-PORN greater than PORN greater than collagen. This response occurred in both media. In addition, the neuritic halo in each instance was markedly more developed in the absence of serum, especially on PNPF-PORN. Nonneuronal behaviors reflected both serum and substratum influences: thus, nonneuronal outgrowth consisted mainly of flat cells with serum and collagen, was nonexistent with serum and PORN or PNPF-PORN, and involved mostly Schwann-like scattered cells in the absence of serum on any one substratum. The serum-dependent behaviors of ganglionic neurites were examined further with explants from chicken E11 sympathetic ganglia. A single substratum was used (PORN), without exogenous trophic factor. Neurite outgrowth was depressed by the presence of fetal calf serum, thus supporting the generality of this phenomenon. Lastly, PC12 cells, a clonal line of rat pheochromocytoma, will grow neurites in the presence of NGF after 48 hr in serum-free, but not serum-containing media. Addition of serum to serum-free cultures at this time results in the rapid and complete retraction of neurites.     

A conditioning lesion enhances sympathetic neurite outgrowth

Axonal regeneration can be influenced by a conditioning lesion (an axonal injury made prior to a second test lesion). Previously, sympathetic neurons in vivo were shown to respond to a conditioning lesion with decreased neurite outgrowth, in contrast to the enhanced outgrowth observed in all other peripheral neurons examined. The present experiments tested the effects of a conditioning lesion on neurite outgrowth in vitro from the superior cervical ganglion (SCG) and the impact of several factors on that response. Ganglia axotomized 1 week earlier and then explanted in Matrigel or collagen gel responded with a significant increase in neurite extension compared to sham-operated ganglia. A distal axotomy produced by unilateral removal of the salivary glands (sialectomy) caused an increase in neurite outgrowth similar to that of a proximal axotomy. These conditioning lesions induced both an increase in the rate of elongation, and, in the case of the proximally axotomized SCG, a shorter initial delay of outgrowth. The enhanced outgrowth following sialectomy was specific to the nerve containing the majority of axons projecting to the salivary glands, suggesting that the conditioning lesion effect is restricted to previously injured neurons. Deletion of the gene for leukemia inhibitory factor (LIF), a gene induced by axotomy, did not abolish the conditioning lesion effect in SCG explants or dissociated cell cultures. In conclusion, sympathetic neurons are capable of responding to a conditioning lesion with increased neurite outgrowth. The hypothesis that the neuronal cell body response to axotomy plays an important role in the conditioning lesion response is discussed.     

脾非淋巴细胞条件培养液作用于颈上节的实验观察

探讨脾非淋巴细胞是否能够产生具有生物活性的神经营养因子。分离ＢＡＬＢ／ｃ小鼠非淋巴细胞并进行原代培养,收集培养上清液作为条件培养液孵育新生小鼠颈上节６０ｈ。结果显示,对对照培养液相比,条件培养液能够地促进体外培养中颈上节交感神经生长的发育,且这一作用在不同程度上可被抗ＮＧＦ和ＮＴ－３抗体特异性阻断。免疫组化染色证实培养的脾非淋巴细胞中抗神经生长因子（ＮＦＧ）和神经营养素－３（ＮＴ－３）免疫反应产物     

The trophic responses of avian sensory ganglia in vitro to N-acetylated and des-acetyl forms of alpha-melanocyte stimulating hormone (alpha-MSH) are qualitatively distinct

alpha-Melanocyte-stimulating hormone (alpha-MSH) accelerates the regrowth of peripheral nerve axons in the rat following their transection (Verhaagen et al., Expl Neurol. 92, 451-454, 1986). The cellular mechanisms of this trophic response were investigated for several naturally occurring derivatives of alpha-MSH using Nerve Growth Factor (NGF)-stimulated quail sensory ganglion explants in vitro in which both neurite outgrowth and non-neuronal cell behaviour could be more reliably observed and quantified. Neurite outgrowth was determined with a semi-quantitative scoring assay. Glial migration into the outgrowth was quantified using a monoclonal antibody, GTE-52, which labels the nuclei of Schwann cells. Des-acetyl alpha-MSH caused a marginal increase in the neurite outgrowth density which was significant at concentrations of 0.04 and 0.1 microgram/ml. The response to acetylated (N-acetyl, N,O-diacetyl) forms of alpha-MSH was characterized by fascicle formation by neurites which resulted in an apparent decrease in the neurite score, and by the outgrowth of non-neuronal cells. Using monoclonal antibody GTE-52, which recognizes a glial nuclear antigen, these cells were identified as Schwann cells. N-Acetyl, but not des-acetyl alpha-MSH increased the number of GTE-52-labelled cells in the NGF-stimulated neurite outgrowth and stimulated their migration in the absence of neurites when NGF was omitted from the culture medium. Exposure of growing explants to two polyclonal antibodies against alpha-MSH resulted in an increased neurite outgrowth density. The results support the hypothesis that alpha-MSH peptides stimulate peripheral nerve growth by modulating the neurite sprouting response, and demonstrate that the nature of the neurotrophic response to naturally occurring melanotropins depends on the existence of acyl substitution at the N-terminal amino acid residue. A possible role of endogenous melanotropin peptides in the regulation of sensory nerve growth is discussed.     

A rapid method for semi-quantitative analysis of neurite outgrowth from chick DRG explants using image analysis

Neurite outgrowth from dorsal root ganglion (DRG) explants is a method of evaluating neurotrophic activity of growth factors and neurotrophin mimetics. The drawbacks to this approach are the difficulties in quantifying the response. Neurite counts are time consuming and labour intensive, and the accuracy is often questionable due to branching and fasciculation of the neurites. We report here a method of semi-quantitative analysis of neurite outgrowth from chick DRG explants, using image analysis to quantify the area occupied by neurites emanating from the ganglion. This method is rapid, takes into account both the length and number of neurites, and is unaffected by neurite fasciculation or branching. Primary explants of chick DRGs were treated with the neurotrophins nerve growth factor (NGF) or neurotrophin-3 (NT-3) and with the compound K252a. K252b was tested for potentiation of the response to NT-3. The results show a dose dependent outgrowth of neurites from explants treated with NGF, NT-3 and K252a, and potentiation of the NT-3 response by K252b. These responses were quantified by neurite area quantification using image analysis. We conclude that neurite area measurement using image analysis provides a robust means of evaluating neurotrophic activity of growth factors and neurotrophin mimetics in vitro.     

p75 and TrkA receptors are both required for uptake of NGF in adult sympathetic neurons: use of a novel fluorescent NGF conjugate

We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red–nerve growth factor (DTR–NGF) conjugate. DTR–NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR–NGF labelling in SCG neurons was shown to be dose-dependent with an EC₅₀ of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR–NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85–93%) reduction in numbers of DTR–NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.     

Extension of sympathetic neurites in vitro towards explants of embryonic and neonatal mouse heart and stomach: ontogeny of neuronotrophic factors

In order to establish when target organs first produce neuronotrophic factors, extension of neurites in vitro from sympathetic ganglia (superior cervical and coeliac) of 1-day neonatal mice towards explants of 10-, 11-, 14- and 17-day embryonic and 1-day neonatal atrium and stomach was examined in co-cultures. Longer neurites extended from ganglia towards, than away from, atrial targets at all stages examined, and was most marked towards 17-day embryonic and neonatal explants. Treatment of atrial co-cultures with antiserum to nerve growth factor (NGF) almost totally blocked preferential neurite outgrowth. Directional growth of neurites towards stomach explants in co-cultures was not as pronounced as that towards atrium; extension of neurites was most marked when stomach was provided by 11-, 14- and 17-day embryos. Such outgrowth was only partially blocked by antiserum to NGF, significant preferential extension of neurites towards stomach persisting in the presence of the antiserum. These results indicate that atrium and stomach produce neuronotrophic factors from the earliest ages studied; the evidence indicates that in the case of atrium, NGF predominates but that stomach produces NGF as well as another factor immunologically distinct from NGF. It is of interest that both types of target explanted before they receive sympathetic innervation show evidence of producing NGF in culture.     

Dose-Dependent Effects of Recombinant Human NGF on Grafted Adult Adrenal Medullary Tissue

It has previously been shown that the chromaffin cells of the adrenal medulla respond to nerve growth factor (NGF) with neurite outgrowth and increased cell survival in tissue culture or after grafting. In the present study we evaluated the dose dependency in neurite outgrowth from chromaffin tissue to recombinant human NGF (rhNGF). Therefore, pieces of adrenal medullary tissue from adult rat were grafted into the anterior chamber of the eye of previously sympathectomized recepients. Survival time was 4 weeks. At grafting and at Days 7, 14, and 21 postgrafting, the eyes were injected with 5 μl of rhNGF at concentrations of 10, 30, 60, 100, 150, and 200 μg/ml, or with a control solution. All grafts, including the controls, survived well and became vascularized. At the low doses of rhNGF, 10 and 30 μg/ml, a small area of the irides was reinnervated and the density of the nerve fiber network was low. The maximal response was obtained at 100 μg rhNGF/ml. Using larger concentrations of 150 and 200 μg rhNGF/ml, the density of the nerve fiber network did not change, but the reinnervated area of the irides was significantly decreased compared to the outgrowth seen in irides treated with 100 μg/ml. In conclusion, adult rat chromaffin tissue responds to rhNGF in a dose-dependent manner. However, at the highest doses used, the outgrowth area was suboptimal, although nerve fiber density was maximal. These results indicate that to obtain maximal effects, the dose of NGF is critical.     

Gangliosides inhibit growth factor-stimulated neurite outgrowth in SH-SY5Y human neuroblastoma cells

Exogenously added gangliosides are known to promote neurite outgrowth in a variety of cell types, including some neuroblastoma cell lines. To study neuritogenesis in SH-SY5Y human neuroblastoma we serum starved the cells for 24 hr and exposed them to gangliosides (GM1, GM3, or GT1b), platelet-derived growth factor (PDGF), insulin, nerve growth factor (NGF), insulin-like growth factor I (IGF-I), or combinations of these for 3 days. We measured four parameters of neurite outgrowth using image analysis. PDGF induced neurite outgrowth in SH-SY5Y and GM1 inhibited this. Both phenomena were dose-dependent with neurites/cell and neurite length being below controls with 100 μM GM1, and percent of neurite-bearing cells being below controls with 25, 50, and 100 μM GM1. Similar but more inhibitory results were obtained with GM3 and GT1b. Insulin and IGF-I induced a neuritogenic response that was less potent than that of PDGF and was also inhibited by gangliosides. NGF had no effect on neurite outgrowth but gangliosides were still inhibitory even in cells not treated with growth factors. From this we conclude that gangliosides inhibit spontaneous and trophic factor-induced neurite outgrowth in SH-SY5Y cells. For GM1 and GT1b, but not GM3, this probably involves inhibition of trophic factor receptor function. J. Neurosci. Res. 47:617–625, 1997. © 1997 Wiley-Liss, Inc.     

NGF/BDNF chimeric proteins: analysis of neurotrophin specificity by homolog-scanning mutagenesis.

Despite their extensive sequence identities at the amino acid level (approximately 55%), NGF and brain-derived neurotrophic factor (BDNF) display distinct neuronal specificity toward neurons of both the PNS and CNS. To explore which region(s) within these neurotrophic factors might determine their differential actions on various subpopulations of peripheral neurons, a systematic series (homolog-scanning mutagenesis) of chimeric NGF/BDNF molecules was prepared using PCR overlap-extension techniques. After expression in COS-7 cells, the chimeric proteins were tested for their biological activities in neurite outgrowth and neuronal survival assays. This approach led to the functional expression of 12 NGF/BDNF chimeras. Surprisingly, despite replacing successive amino acid segments throughout the entire length of NGF with the corresponding parts of BDNF, all chimeras displayed full NGF-like activity in bioassays carried out with PC12 cells, embryonic chick dorsal root ganglion explants, sympathetic ganglion explants, and dissociated cultures of dorsal root ganglion neurons. Most of the chimeras additionally showed BDNF-like activity as defined by neurite outgrowth on chick nodose ganglion explants. However, none of the chimeras supported the survival of dissociated nodose ganglion neurons. Our results suggest that NGF and BDNF must share very similar higher-order protein structures, and we propose that the overall structure or conformation of NGF, in contrast to short amino acid "active-site" segments, may determine its exact neuronal specificity.     

Rat alpha(2)-macroglobulin inhibits NGF-promoted neurite outgrowth, TrK phosphorylation, and gene expression of pheochromocytoma PC12 cells.

Rat alpha-1-macroglobulin (alpha(1)M) and alpha-2-macroglobulin (alpha(2)M) are murine homologs of human alpha(2)M, and rat alpha(2)M is generally known as an acute-phase protein. Monoamine-activated forms of human alpha(2)M have been shown to inhibit various neuronal functions, but the effect of rat alpha(1)M and acute-phase alpha(2)M on neurons is largely unknown. In this report, rat serotonin-activated alpha(2)M (5HT-alpha(2)M) has been demonstrated to inhibit nerve growth factor (NGF)-promoted neurite extension in pheochromocytoma PC12 cells, and we investigated its possible mechanism of action including its effect on NGF-promoted signal transduction and gene expression in these cells. Especially in the absence of NGF, 5HT-alpha(2)M was found to bind to TrkA (the high-affinity receptor for NGF) much better than normal alpha(2)M (N-alpha(2)M). 5HT-alpha(2)M dose-dependently inhibited NGF-promoted autophosphorylation of TrkA, and decreased the expression of two immediate-early genes (NGFI-A and c-jun) and two delayed-response genes (SCG10 and transin) which are associated with neurite outgrowth in PC12 cells. The unmodified N-alpha(2)M, on the other hand, exhibited very little or no inhibitory effects on neurite extension, Trk phosphorylation, or expression of these genes. The results of this study taken together suggest that monoamine-activated acute-phase rat alpha(2)M appears to inhibit neurite outgrowth in PC12 cells possibly via its direct binding to TrkA and subsequent blocking of TrkA-mediated signal transduction and gene expression.