Influence of soluble environmental factors on the development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium: comparison with the effects of L-triiodothyronine (L-T3)

In cerebral hemisphere neuronal cultures derived from 15-day-old rat embryos, the addition of L-triiodothyronine (L-T3) or nerve growth factor (NGF) enhanced the expression of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in a dose-dependent manner. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT and AChE activities was significantly greater than the sum of the individual effects. Conversely, when the cultures were exposed to astrocyte conditioned medium grown in the presence or absence of L-T3 (CM + L-T3 or CM-L-T3). laminin and fibroblast growth factor (FGF), ChAT and AChE activities were not stimulated above those of control cultures when added alone or in combination with L-T3. Furthermore, L-T3, NGF, CMs, laminin and FGF did not affect AChE+ cell survival, but significantly increased neurite outgrowth and branching with NGF and L-T3 being the most powerful agents followed by CMs, laminin and FGF. Additionally, the simultaneous addition of L-T3 with either laminin or FGF in culture, caused an additive effect of L-T2 in the neurite density of AChE+ cells with both agents. This study shows that (1) thyroid hormones do not act through the regulation of soluble neurotrophic factors produced by astroglial cells, (2) thyroid hormones interact with the effect of NGF on ChAT and AChE activities, (3) the regulation of ChAT and AChE activities and the neurite outgrowth are independently regulated. and (4) the regulation of ChAT and AChE activities is very specific compared with that of neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of axonal growth from sensory neurons by excess nerve growth factor

Fifteen-day embryonic rat dorsal root ganglion (DRG) neurons were exposed to 1 to 200 ng/ml nerve growth factor (NFG). Maximal neurite outgrowth was obtained with 10 to 20 ng/ml. Neurite outgrowth was reduced to 89% of maximal by increasing NGF to 50 ng/ml, to 66% by 100 ng/ml, and to 18% by 200 ng/ml NGF. Identical effects were seen with mouse 2.5S NGF and recombinant human NGF. Neuron cell counts demonstrated that significant cell death did not occur. In time course experiments, significant inhibition, compared with control, began within 1 hour of adding 200 ng/ml and 3 hours of adding 50 ng/ml NGF. The inhibitory effect of NGF on neurite outgrowth was reversed within 3 hours when DRG were incubated with 5 ng/ml NGF after treatment with 50 or 200 ng/ml NGF medium for 12 hours. The inhibition demonstrated for neurons did not occur in PC12 cells; axonal growth was not inhibited by up to 1,000 ng/ml NGF. Excess brain-derived neurotrophic factor or neurotrophin-3 did not inhibit neurite outgrowth. We conclude that high concentrations of NGF produces specific and reversible arrest of neurite outgrowth from sensory neurons. This observation has important clinical implications, because these inhibitory concentrations have been exceeded when NGF has been administered into the central nervous system of humans and animals.     

Cooperation between nerve growth factor and laminin or fibronectin in promoting sensory neuron survival and neurite outgrowth

Chick embryo dorsal root ganglion (DRG) neurons were purified by differential adhesion to plastic. The purified neurons were used to study the cooperation between nerve growth factor (NGF) and laminin or fibronectin in promoting neuron survival and neurite outgrowth. NGF alone supported the survival of only 20% embryonic day 10 (E10) cells, of which only 40-50% had neurites. Treatment of the substrate with fibronectin or laminin increased survival in the presence of NGF up to 80% of the seeded neurons, all of which showed extensive neurite outgrowth. Survival and neurite outgrowth were also enhanced by the combined effects of elevated potassium and laminin. In contrast to E8-10 cells, 85% of E16 neurons survived in the basal culture conditions, i.e. without additional NGF, fibronectin or laminin, although neurite outgrowth was enhanced by all 3 proteins. Antisera to NGF, laminin and fibronectin, each independently decreased survival and neurite outgrowth of DRG neurons, totally with E9 and partially with E16 cells. The results suggest that the cooperative actions of extracellular matrix proteins and NGF are essential for survival and neurite outgrowth of embryonic DRG neurons and that these neuronal requirements change during development.     

Evidence that the B2 chain of laminin is responsible for the neurite outgrowth-promoting activity of astrocyte extracellular matrix.

Extracellular matrix (ECM) derived from cerebral cortical astrocytes stimulates neurite outgrowth from pheochromocytoma (PC12) cells in the absence of the classical nerve growth factor (NGF). We have shown here that astrocyte ECM can also stimulate neurite outgrowth from primary cultures of central nervous system (CNS) neurons. Using PC12 cells for a quantitative assay, we also demonstrated that the neurite growth-promoting activity increased as the astrocytes matured in vitro: ECM from older astrocytes (3-12 weeks in vitro) exhibited two-fold more neurite growth-promoting activity than ECM for younger astrocytes (5 days to 2 weeks in vitro). We applied various antibodies to identify the neurite growth-promoting factor of astrocyte ECM and found that anti-laminin inhibited neurite outgrowth by 50%, whereas anti-fibronectin and anti-NGF had no effect. Immunoblots, using laminin chain-specific antibodies, and cDNA hybridization of laminin mRNA demonstrated that cultured astrocytes synthesize only the B2 chain of laminin. This suggests that the B2 chain of laminin suffices to stimulate neurite outgrowth.     

Effects of IL-1beta, IL-6 or LIF on rat sensory neurons co-cultured with fibroblast-like cells.

Inflammation may affect the local presence of sensory nerve fibers in situ and inflammatory mediators influence sensory neurons in vitro. In the present study we have investigated effects of the cytokines interleukin-1beta (IL-1beta, interleukin-6 (IL-6), and leukemia inhibitory factor (LIF) on survival of and neurite growth from neonatal rat sensory neurons co-cultured with fibroblast-like cells prepared from neonatal rat skin (sFLCs) or perichondrium (pFLCs). The results showed that both FLC types expressed receptors for all three cytokines. Five ng/ml of either cytokine, but not lower or higher concentrations, supported survival of DRG neurons co-cultured with sFLCs. Neuronal survival was also enhanced by addition of the soluble IL-6 receptor (rsIL-6R) with or without IL-6. In co-cultures with pFLCs neuronal survival was promoted by IL-6, increasing with cytokine concentration. Addition of rsIL-6R without IL-6 did also stimulate neuronal survival. The growth of neurites from DRG neurons co-cultured with sFLCs was stimulated by 0.5 ng/ml LIF, unaffected by 5 ng/ml LIF and inhibited by 50 ng/ml LIF. Considering DRG neurons co-cultured with pFLCs, 50 ng/ml of either of the three cytokines, as well as rsIL-6R conditioned medium, stimulated neurite outgrowth. Some of the cytokine effects observed were reduced by application of antibodies against nerve growth factor (NGF). We conclude that that the cytokines examined affect DRG neurons in terms of survival or neuritogenesis, that the effects are influenced by cytokine concentration and the origin of the FLCs and that some of the effects are indirect, probably being mediated by factors released from FLCs.     

Depletion of a fatty acid-binding protein impairs neurite outgrowth in PC12 cells

Epithelial fatty acid-binding protein (E-FABP) is up-regulated in rat dorsal root ganglia after sciatic nerve crush and in differentiating neurons during development. The present study investigates the role of E-FABP during nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells. Undifferentiated PC12 cells express low levels of E-FABP, while NGF triggers a 6- and 8-fold induction of E-FABP mRNA and protein, respectively. Up-regulation of E-FABP mRNA occurs as early as 24 h after NGF treatment and remains highly expressed over the course of several days, corresponding to NGF-mediated neurite outgrowth. Withdrawal of NGF leads to down-regulation of E-FABP mRNA and retraction of neurites. Immunofluorescence microscopy reveals E-FABP immunoreactivity in the perinuclear cytoplasm, neurites and growth cones of NGF-differentiated cells. To examine the role of E-FABP during neurite outgrowth, PC12 cells were transfected with a constitutive antisense E-FABP vector to create the E-FABP-deficient line PC12-AS. By morphometric analysis, PC12-AS cells treated for 2, 4, and 7 days with NGF exhibited significantly decreased neurite expression relative to control (mock-transfected) cells. Taken together, these data indicate that E-FABP is important in normal NGF-mediated neurite outgrowth in PC12 cells, a finding that is consistent with a potential role in axonal development and regeneration.     

Purine analogs inhibit nerve growth factor-promoted neurite outgrowth by sympathetic and sensory neurons

Past studies have shown that purine analogs block certain, but not all, responses of cultured rat PC12 pheochromocytoma cells to nerve growth factor (NGF). In the present work, newborn rat sympathetic and sensory neurons were exposed to NGF in the presence or absence of the purine analogs 6-thioguanine and 2-aminopurine. These compounds reversibly suppressed NGF-dependent neurite outgrowth by the neurons and did so at concentrations comparable to those effective on PC12 cells. In contrast to their effects on neurites, neither compound significantly blocked NGF-promoted neuronal survival. Similar effects were seen with cultures of chick embryo sympathetic ganglia. These findings show that purine analog effects on NGF responses can be extended to mammalian and avian neurons. Moreover, the differential effects of the analogs on neurite outgrowth and survival indicate that these 2 actions of NGF can be dissected from one another and may represent different mechanistic pathways.     

Acetyl-L-carnitine enhances the response of PC12 cells to nerve growth factor.

We have demonstrated that treatment of rat pheochromocytoma (PC12) cells with acetyl-L-carnitine (ALCAR) stimulates the synthesis of nerve growth factor receptors (NGFR). ALCAR has also been reported to prevent some age-related impairments of the central nervous system (CNS). In particular, ALCAR reduces the loss of NGFR in the hippocampus and basal forebrain of aged rodents. On these bases, a study on the effect of NGF on the PC12 cells was carried out to ascertain whether ALCAR induction of NGFR resulted in an enhancement of NGF action. Treatment of PC12 cells for 6 days with ALCAR (10 mM) stimulated [125I]NGF PC12 cell uptake, consistent with increased NGFR levels. Also, neurite outgrowth elicited in PC12 cells by NGF (100 ng/ml) was greatly augmented by ALCAR pretreatment. When PC12 cells were treated with 10 mM ALCAR and then exposed to NGF (1 ng/ml), an NGF concentration that is insufficient to elicit neurite outgrowth under these conditions, there was an ALCAR effect on neurite outgrowth. The concentration of NGF necessary for survival of serum-deprived PC12 cells was 100-fold lower for ALCAR-treated cells as compared to controls. The minimal effective dose of ALCAR here was between 0.1 and 0.5 mM. This is similar to the reported minimal concentration of ALCAR that stimulates the synthesis of NGFR in these cells. The data here presented indicate that one mechanism by which ALCAR rescues aged neurons may be by increasing their responsiveness to neuronotrophic factors in the CNS.     

Nerve growth factor and a fibroblast growth factor-like neurotrophic activity in cerebrospinal fluid of brain injured human patients

We report here the presence of nerve growth factor (NGF) in the cerebrospinal fluid (CSF) of some brain-injured human patients soon after injury. The NGF was quantified against a recombinant human NGF standard in a two-site enzyme-linked immunoabsorbant assay using antibodies against murine B NGF. None of the samples collected more than 2 days after injury contained detectable levels of NGF. When the CSF was assayed for the ability to promote neurite outgrowth from PC12 cells, neurite outgrowth was reduced, but not completely blocked, by antibodies to B NGF, suggesting that there were other biologically active factors present. Fibroblast growth factor (FGF) also promotes neurite outgrowth in PC12 cells. In an initial screening for the presence of FGF, we employed PC12 cells and NR119 cells, PC12 variants in which recombinant human B NGF, but not recombinant human basic FGF, promotes neurite outgrowth. CSF from brain injury patients promoted greater neurite outgrowth from PC12 cells than from NR119 cells, suggesting that some of the biological activity associated with the injury CSF may be due a FGF. This possibility is further supported by the observation that the biological activity of the injury CSF significantly reduced by batch absorption with heparin Sepharose, suggesting the presence of a heparin binding neurotrophic factor. Neurotrophic factors appear in CSF as a consequence of diverse types of brain injury, including head trauma, intracerebral hemorrhage and subarachnoid hemorrhage. The appearance of these factors may reflect important common elements in the complex series of cellular changes occuring in response to acute brain injury.     

Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. © 1994 Wiley-Liss, Inc.     

Ethanol enhances growth factor-induced neurite formation in PC12 cells.

Ethanol can injure the nervous system by disturbing the growth of neural processes. PC12 cells, which form neurites in response to nerve growth factor (NGF), fibroblast growth factor (FGF), and cAMP analogues, were used to study mechanisms by which ethanol alters process outgrowth. Ethanol potentiated NGF-induced neurite outgrowth in cells cultured on different substrata and in serum-containing or defined medium. Ethanol did not increase NGF receptor binding or internalization of NGF. Neurite outgrowth induced by basic FGF was also increased by ethanol but outgrowth induced by forskolin was not. Ethanol potentiated NGF-induced expression of Thy-1, but not of neural cell adhesion molecule (N-CAM), indicating that some, but not all actions of NGF are enhanced by ethanol. In some brain regions, chronic exposure to ethanol increases the growth of dendrites. This has been explained as a compensatory response of surviving neurons to the loss of neighboring cells, and not as a direct effect of ethanol. The present findings suggest that, in some cells, ethanol directly promotes growth factor-mediated neurite formation. This could harm the nervous system by disturbing the balanced development and organization of synapses.     

Differential effects of cyclic AMP and cholera toxin on nerve growth factor-induced neurite outgrowth from adrenal medullary chromaffin and pheochromocytoma cells

The extension of neurites from adrenal medullary chromaffin cells and PC 12 cells upon addition of nerve growth factor (NGF) has been proposed to be mediated by cyclic AMP. It is shown here that substances increasing intracellular cyclic AMP levels have a reverse effect on NGF-induced neurite outgrowth of these two related cell types. Hence, cyclic AMP is not generally involved in neurite outgrowth from NGF responsive cells. Furthermore, it is concluded that PC 12 cells cannot always be considered as a suitable model for adrenal medullary chromaffin cells.     

NGF induces neurite outgrowth via a decrease in phosphorylation of myosin light chain in PC12 cells.

The relationship between phosphorylation of myosin light chain (MLC) and neurite outgrowth induced by nerve growth factor (NGF) was studied in PC12 cells. Inhibitors of Rho kinase, HA-1077 or Y-27632 also induced neurite outgrowth. As already reported botulinum exoenzyme C3 which inactivates Rho protein also induced neurite outgrowth. Calyeulin A, an inhibitor of phosphatase counteracted both NGF- and C3- induced neurite outgrowth. Treatments of both NGF and C3 resulted in significant and transient decrease in phosphorylated MLC. These results suggest that NGF induces neurite outgrowth of PC12 by a transient decrease in phosphorylated MLC which is brought about by activation of MLC phosphatase via inhibition of Rho-Rho kinase pathway.     

p21(ras) stimulates pathways in addition to ERK, p38, and Akt to induce elongation of neurites in PC12 cells

Initiation and elongation of neurites in PC12 cells has been shown to be stimulated by nerve growth factor (NGF). Initiation of NGF-stimulated neurites in a PC12 subclone (PC12-N09) is rapid, giving rise to short neurites that do not elongate after 1 day. To determine whether increasing activation of p21(ras) could restore neurite elongation in these cells and whether it would affect the phosphorylation of signaling proteins, the subclone PC12-N09 was transfected with constitutively active p21(ras61L) (PC12-N09ras61L) and neurite outgrowth with or without NGF was determined. Overexpression of wild-type p21(ras) (PC12-N09rasWT) did not lead to spontaneous neurite initiation but restored the ability of NGF to stimulate continuous neurite elongation. However, NGF-stimulated phosphorylation of ERK, p38, and Akt in PC12-N09rasWT cells is similar in duration to that in PC12-N09 cells, indicating that the p21(ras) signaling through ERK, p38, and Akt was not involved in the restoration of normal neurite elongation in PC12-N09 cells. These results show that p21(ras)-activated pathways other than ERK, p38, and Akt are necessary for appropriate NGF-stimulated neurite elongation in PC12 cells.     

Heat shock induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway

We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.     

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells

Dental pulp stem cells (DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium (DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor (NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2 (MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction (qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors (NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCs-CM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls; however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

Chinese Library Classification No. R459.9; R364; R622