Heterogeneity of human neuroblastoma cell lines in their proliferative responses to basic FGF,NGF, and EGF: Correlation with expression of growth factors and growth factor receptors

Growth factors can induce both proliferation or differentiation of neuroblastoma (NB) cells through interaction with specific receptors. Using two automated colorimetric assays for determinations of cell numbers, the present study demonstrates that (a) different NB and neuroepithelioma cell lines show distinct responses, both qualitatively and quantitatively, to basic FGF (bFGF), NGF, and EGF(b) even closely related NB cell lines (e.g., SK-N-SH, SH-SY5Y, and SHEP) do not respond uniformly to these factors; c) responses of the two neuroepithelioma cell lines employed (SK-N-MC and CHP-100) differ, but match those of certain NB cell lines; and d) two growth factors, bFGF and EGF, may both stimulate or inhibit proliferation, depending on the cell line studied. Specifically, IMR-32, SK-N-SH, and SH-SY5Y showed a mitogenic response to each growth factor. Maximal proliferative responses ranged from 204–355% as compared to controls (100%). GICAN was stimulated by NGF (199%), and SK-N-MC and NMB by EGF (282 and 140%, respectively), but other factors were ineffective. CHP-100 and GIMEN were inhibited by bFGF. NGF and EGF were not effective on CHP-100 cells, while EGF caused an arrest of mitogenic activity in GIMEN cells, and NGF stimulated their proliferation. Cell lines SHEP and LAN1 did not respond to any factor. To begin to analyze putative relationships of growth factor responsiveness and growth factor/growth factor receptor expressions, IMR-32, GIMEN, and LAN1 cell lines were studied for the presence of bFGF, NGF, FGF receptors (R)-1 (flg) and FGFR-4, trk, and low-affinity NGF receptor (p75) mRNAs. All three cell lines expressed bFGF and NGF mRNA, but not the FGFR-1, FGFR-4, trk, and p75 mRNAs. These results suggest extremely diverse patterns of NB/neuroepithelioma cell responsiveness to “mitogenic” growth factors and no overt correlation between such responses and growth factor/growth factor receptor expression. © 1995 Wiley-Liss, Inc.     

Retroviral transfer of the beta-nerve growth factor gene into murine neuroectodermal tumor cells modulates cell proliferation rate, neurite formation, and NGF binding site expression

The response of wild-type and genetically engineered neuroectodermal tumor (NET) cells to exogenous and endogenously synthesized nerve growth factor (NGF) was investigated. Differences in cell proliferation rate, neurite formation, and expression of NGF binding sites were quantitatively determined. Ecotropic retroviral vectors were used to transfer the genes for beta-galactosidase (beta-GAL) and NGF into wild-type C-1300 and Neuro-2A murine neuroblastoma (MNB) and rat pheochromocytoma (PC-12) cells. Conditioned media obtained from NET cells infected with the NGF gene contained biologically active NGF, whereas media from beta-GAL infected cells did not. Infection with the NGF vector induced a short-term decrease in cell proliferation rate and increased neurite formation in wild-type, substrate-adherent PC-12 and Neuro-2A MNB cells (P > 0.05). Incubation of wild-type C-1300, Neuro-2A MNB, and PC-12 cells with NGF (0-200 ng/ml) for 5 days significantly reduced proliferation rates in a concentration-dependent manner and increased neurite extrusion. All NGF-NET cells had a significantly diminished response to the antiproliferative action of exogenous NGF. Ligand binding assays with 125I-NGF demonstrated a marked reduction in the number of NGF binding sites on NGF-NET cells compared to wild type. The attenuated response of NGF-NET cells to exogenous NGF correlated positively with the down-regulation of NGF binding sites. In conclusion, beta-NGF gene transfer into wild-type NET cells induces the synthesis and secretion of NGF, temporarily decreases cell proliferation rate, increases neurite extrusion, down-regulates NGF binding sites, and reduces NET cell responsiveness to NGF. A putative role for NGF may be the modulation of NET cell proliferation and differentiation.     

Specificity of nerve growth factor signaling: Differential patterns of early tyrosine phosphorylation events induced by NGF,EGF, and bFGF

The specificity of nerve growth factor (NGF) action was examined by comparing early tyrosine phosphorylation events induced by NGF, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF). In PC12 cells, administration of either the differentiation factor NGF or the mitogenic factor EGF led to tyrosine phosphorylation of multiple polypeptides in the 100–110 kDa size range associated with PI-3 kinase. However, NGF induced a more prolonged phosphorylation, relative to a transient EGF effect. In contrast, the differentiation factor bFGF failed to induce measurable tyrosine phosphorylation of PI-3 kinase-associated proteins. Similarly, NGF but not bFGF induced marked tyrosine phosphorylation of PLCγ, another early signaling molecule, suggesting that multiple pathways exist for promoting differentiation, and/or that these signaling molecules are not essential for differentiation. TrkA signaling was also compared between PC 12 cells and NIH-3T3 cells heterologously expressing trkA, where receptor activation promotes mitogenesis. In this comparison, significant differences were observed in the tyrosine phosphorylation pattern of PI-3 kinase-associated polypeptides, suggesting the existence of cell type-specific molecular interactions influencing trkA signaling. Mechanistically, NGF stimulation of PC12 cells resulted in a weak or possibly indirect association between trkA and PI-3 kinase. Furthermore, NGF did not appear to activate or substantially alter the overall level of PI-3 kinase activity, raising the possibility that ligand-induced phosphorylation may serve instead to relocalize constitutively active PI-3 kinase molecules within the cell. Taken together, data presented suggest that the temporal pattern of induced phosphorylation, the nature of induced associations with other phosphoproteins, and cell type-specific components may all contribute to the generation of NGF signaling specificity. © 1995 Wiley-Liss, Inc.     

Effect of Single Growth Factor and Growth Factor Combinations on Differentiation of Neural Stem Cells

Objective

The effects on neural proliferation and differentiation of neural stem cells (NSC) of basic fibroblast growth factor-2 (bFGF), insulin growth factor-I (IGF-I), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) were assessed. Also, following combinations of various factors were investigated : bFGF+IGF-I, bFGF+BDNF, bFGF+NGF, IGF-I+BDNF, IGF-I+NGF, and BDNF+NGF.

Methods

Isolated NSC of Fisher 344 rats were cultured with individual growth factors, combinations of factors, and no growth factor (control) for 14 days. A proportion of neurons was analyzed using β-tubulin III and NeuN as neural markers.

Results

Neural differentiations in the presence of individual growth factors for β-tubulin III-positive cells were : BDNF, 35.3%; IGF-I, 30.9%; bFGF, 18.1%; and NGF, 15.1%, and for NeuN-positive cells was : BDNF, 34.3%; bFGF, 32.2%; IGF-1, 26.6%; and NGF, 24.9%. However, neural differentiations in the absence of growth factor was only 2.6% for β-tubulin III and 3.1% for NeuN. For β-tubulin III-positive cells, neural differentiations were evident for the growth factor combinations as follows : bFGF+IGF-I, 73.1%; bFGF+NGF, 65.4%; bFGF+BDNF, 58.7%; BDNF+IGF-I, 52.2%; NGF+IGF-I, 40.6%; and BDNF+NGF, 40.0%. For NeuN-positive cells : bFGF+IGF-I, 81.9%; bFGF+NGF, 63.5%; bFGF+BDNF, 62.8%; NGF+IGF-I, 62.3%; BDNF+NGF, 56.3%; and BDNF+IGF-I, 46.0%. Significant differences in neural differentiation were evident for single growth factor and combination of growth factors respectively (p<0.05).

Conclusion

Combinations of growth factors have an additive effect on neural differentiation. The most prominent neural differentiation results from growth factor combinations involving bFGF and IGF-I. These findings suggest that the combination of a mitogenic action of bFGF and postmitotic differentiation action of IGF-I synergistically affects neural proliferation and NSC differentiation.     

Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, epidermal growth factor, insulin and the insulin-like growth factors I and II

To study the selectivity of neurotrophic actions in the brain, we analyzed the actions of several known growth factors on septal cholinergic, pontine cholinergic, and mesencephalic dopaminergic neurons in culture. Similar to nerve growth factor (NGF), basic fibroblast growth factor (bFGF) stimulated choline acetyltransferase activity in septal cultures. In contrast to NGF, bFGF also enhanced dopamine uptake in mesencephalic cultures and stimulated cell proliferation in all 3 culture types. Insulin and the insulin-like growth factors I and II stimulated transmitter-specific differentiation and cell proliferation in all culture types. Epidermal growth factor (EGF) produced a small increase in dopamine uptake by mesencephalic cells and stimulated cell proliferation in all culture types. In septal cultures, bFGF was most effective when given at early culture times, NGF at later times. The stimulatory actions of bFGF and insulin did not require the presence of glial cells and were not mediated by NGF. In mesencephalic cultures, the stimulation of dopamine uptake by bFGF and EGF was dependent on glial proliferation. The results suggest different degrees of selectivity of the neurotrophic molecules. NGF and, very similarly, bFGF seem to influence septal cholinergic neurons directly and rather selectively, whereas the neurotrophic actions of insulin and the insulin-like growth factors appear to be more general.     

PC12h-R cell,a subclone of PC12 cells,shows EGF-induced neuronal differentiation and sustained signaling

Unlike nerve growth factor (NGF), epidermal growth factor (EGF) does not induce neuronal differentiation but promotes proliferation of the rat pheochromocytoma PC12 cells. We found that PC12h-R, a subclone of PC12 cells, differentiated into neuron-like cells in response to EGF as well as to NGF. PC12h-R cells treated with EGF extended neurites, attenuated cell proliferation, and increased the levels of tyrosine hydroxylase protein synthesis and of acetylcholinesterase activity as those treated with NGF. The EGF-induced differentiation of PC12h-R cells was not mediated by the indirect activation of p140^trkA by EGF. In addition, EGF induced the sustained tyrosine phosphorylation of the EGF receptor, mitogen-activated protein (MAP) kinases, and 46 and 52 kDa proteins, and the prolonged activation of MAP kinases in PC12h-R cells compared with the parent PC12h, which does not show EGF-induced differentiation. The response of PC12h-R cells to EGF was not simply due to an increase in the level of EGF receptor protein. These results indicated that the duration of EGF-induced signaling might determine the cellular response of PC12 cells between cell proliferation and neuronal differentiation. © 1996 Wiley-Liss, Inc.     

Effects of growth factors on phosphatidylinositol-3 kinase in astroglial cells

Growth factors differently regulate astroglial cell differentiation and proliferation. In an effort to understand the early intracellular events promoted by growth factors in astroglial cells, we have determined the effects of insulin-like growth factor I (IGF1), insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factors (FGFs) on phosphatidylinositol-3 kinase (PI(3)-kinase). In astroglial cells cultured in serum-free medium, IGF1, PDGF, and EGF, which stimulate cell proliferation, increased PI(3)-kinase activity immunoprecipitated with anti-phosphotyrosine antibodies as shown by thin layer chromatography and high performance liquid chromatography. FGFa and FGFb, which strongly stimulate proliferation, glutamine synthetase, and deiodinase activities and modify cell morphology, have no effect on PI(3)-kinase activity. Addition of 1 nM PDGF, 10 nM IGF1, or 100 nM EGF to the culture medium rapidly stimulated PI(3)-kinase activity which declined slowly after 2 min. The stimulation of PI(3)-kinase increased with growth factor concentration. The maximum increase in PI(3)-kinase activity occurred with 50 nM IGF1, 1 nM PDGF, or 100 nM EGF. Since insulin was active only at high concentration (1 μM), its effect was probably mediated through IGF1 receptors and not through IGF1 receptors and not through insulin receptors. IGF1 and PDGF, to a lesser degree, also increased the PI(3)-kinase activity associated with pp60^c-src protein. Immunoblots performed with an antibody directed against the p⁸⁵-subunit of the PI(3)-kinase confirmed that IGF1 increased the number of PI(3)-kinase molecules associated with phosphotyrosine-containing proteins or with c-src protein. Each growth factor affects in a different manner the association of PI(3)-kinase with phosphotyrosine-containing proteins and with pp60^c-src and thus probably modulates intracellular signals downstream of PI(3)-kinase in astroglial cells. © 1995 Wiley-Liss, Inc.     

Growth factor-mediated neural proliferation: target of ethanol toxicity

Ethanol exposure during development is teratogenic. The central nervous system (CNS) is particularly susceptible to ethanol toxicity. In fact, heavy gestational ethanol consumption is one of the leading known causes of mental retardation in the Western world. Ethanol exposure disrupts the proliferation of glia and neuronal precursors in the developing CNS. Depending upon cell population and blood ethanol concentration, ethanol can either inhibit or stimulate cell proliferation. Two features of cell proliferation that are affected by ethanol exposure are the growth fraction (the proportion of cells that is actively cycling) and the cell cycle kinetics, particularly in the length of the G1 phase of the cell cycle. Cell proliferation in the developing CNS reflects the action of positive (mitogenic growth factors) and negative (anti-proliferative factors) regulators. Increasing evidence shows that ethanol interferes with the action of growth factors. In vitro systems are a good model to investigate ethanol neurotoxicity, since the effects of ethanol on cultured cells parallel the effects of ethanol in the developing CNS. The inhibitory effects of ethanol on cell proliferation may result from interference with mitogenic growth factors (e.g., bFGF, EGF, PDGF, IGF-I). Conversely, the stimulatory effects of ethanol may result from the interference with growth inhibiting factors (e.g., TGFβ1). Interestingly, both in vivo and in vitro studies show that proliferating neural cells display differential sensitivity to ethanol. This differential sensitivity correlates with their response to mitogenic growth factors; that is, cells that are actively regulated by mitogenic growth factors are much more susceptible to ethanol than cells that are less or unresponsive to such factors. Ethanol interference with growth factor action could occur at three levels: ligand production, receptor expression, and/or signal transduction. Thus, ethanol-induced alterations in the developing CNS that characterize fetal alcohol syndrome apparently result from alterations in the regulatory action of growth factors.     

Differential effects of basic fibroblast growth factor,epidermal growth factor,transforming growth factor-α, and insulin-like growth factor-I on a hypothalamic gonadotropin-releasing hormone neuronal cell line

Recent studies in several neuronal lineages suggest that extrinsic factors such as polypeptide growth factors regulate various stages of neuronal development, from initial commitment of multipotent progenitors to induction of specific gene expression that is characteristic of terminal neuronal differentiation. In the present study, immortalized hypothalamic neurons of the GT1-1 lineage were used to analyze proliferative, as well as morphological and molecular differentiation actions of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and insulin-like growth factor-I (IGF-I). These effects were compared with those induced by specific activators of protein kinase A and C pathways, which potently inhibited cell proliferation and gonadotropin-releasing hormone (GnRH) gene expression, but stimulated morphological neuronal maturation as determined by the length and number of neurite outgrowth. bFGF exerted a broad spectrum of stimulatory effects, increasing the rate of proliferation measured both by the incorporation of ³H-thymidine and by cell number, and parameters of terminal differentiation, such as neurite outgrowth and induction of gene expression. bFGF stimulated the expression of the hybrid transgene-containing portions of the rat GnRH promoter. In contrast, EGF, TGF-α, and IGF-I inhibited cell proliferation, while having subtle effects on neurite outgrowth. Thus, GT1-1 cells appear to be differentially responsive to distinct neurotrophic factors, providing a model for studying the specific effects of neurotrophic factors on functional differentiation, migration, and connectivity of hypothalamic neurons. J. Neurosci. Res. 49:739–749, 1997. © 1997 Wiley-Liss, Inc.     

Epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and basic fibroblast growth factor (bFGF) differentially influence neural precursor cells of mouse embryonic mesencephalon

Growth factors are key elements in the process of neural cell differentiation. We examined the effects of classical mitogens on neural precursor cells, by culturing mouse cells of the embryonic (13.5 days postcoitum) mesencephalon and treating them with epidermal growth factor (EGF), transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and transforming growth factor-β (TGF- β). Our initial results show that EGF, TGF-α or bFGF, but not NGF or TGF-β, induced general proliferation of the cultured cells, followed by formation of colonies. Combinations of these three growth factors suggest that most cells with the capacity to form colonies responded to EGF, TGF-α, or bFGF. The number of colonies increased significantly when EGF, but not TGF-α, was used in combination with bFGF. Furthermore, a population responding only to EGF + bFGF was detected in the dorsal mesencephalon. The colony-forming activity of bFGF was dependent on insulin, but bFGF and insulin cooperation was indirect since we could not observe colony formation in subcultures of cells derived from colonies, even in the presence of insulin. Cells obtained from our colonies displayed neuronal and glial morphology and expressed markers of both neurons and astrocytes; nestin, a marker of neural precursor cells was also expressed in the majority of colonies. Growth factors also influenced neuronal maturation; the best neurite outgrowth was obtained from cells derived from bFGF-induced colonies cultured in the presence of EGF + bFGF. These data indicate the existence of neural precursor cells in the embryonic mesencephalon that respond differentially to growth factors. © 1995 Wiley-Liss, Inc.     

Neurite growth promotion by nerve growth factor and insulin-like growth factor-1 in cultured adult sensory neurons: role of phosphoinositide 3-kinase and mitogen activated protein kinase

Although neurons of the PNS no longer require neurotrophins such as Nerve Growth Factor (NGF) for their survival, such factors are involved in regulating axonal sprouting and regeneration after injury. In addition to the neurotrophin receptors, sensory neurons are reported to express IGF-1, EGF and FGF receptors. To investigate the influence of growth factors in addition to NGF, we examined the effects of IGF-1 EGF and FGF on neurite growth from adult rat dorsal root ganglion sensory neurons in both dissociated cultures and in compartmented cultures. As expected, NGF elicited robust neuritic growth in both the dissociated and compartmented cultures. The growth response to IGF-1 was similar, although there was minimal neurite growth in response to EGF or FGF. In addition, IGF-1 (but neither FGF nor EGF), when applied to cell bodies in compartmented cultures, potentiated the distal neurite growth into NGF-containing side compartments. This potentiation was not seen when these factors were provided along with NGF in the side compartments of compartmented cultures, or in the dissociated cultures. To determine the contribution of signaling intermediates downstream of receptor activation, we used inhibitors of the potential effectors and Western blotting. The PI 3-kinase inhibitor, LY294002 attenuated neurite growth evoked by NGF, IGF and EGF in dissociated cultures, although the MAP kinase kinase (MEK) inhibitor PD098059 diminished the growth in only IGF. Immunoprecipitation and Western blotting results demonstrated differential activation of MAPK, PI 3-kinase, PLCgamma1 and SNT by the different factors. Activation of PI 3-kinase and SNT by both NGF and IGF-1 correlated with their effects on neurite growth. These results support the hypothesis that the PI 3-kinase pathway plays an important role in neuritogenesis.     

Modulation of extracellular signal‐related kinase,cyclin D1, glial fibrillary acidic protein,and vimentin expression in estradiol‐pretreated astrocyte cultures treated with competence and progression growth factors

The present study seeks to elucidate the interactions between the “competence” growth factor basic fibroblast growth factor (bFGF) and/or estrogen 17β‐estradiol and the “progression” growth factors epidermal growth factor (EGF), insulin‐like growth factor‐I (IGF‐I), and insulin (INS) on DNA labeling and also cyclin D1, extracellular signal‐related kinase 1/2 (ERK1/2), glial fibrillary acidic protein (GFAP), and vimentin expression in astroglial cultures under different experimental conditions. Pretreatment for 24 hr with bFGF and subsequent exposure for 36 hr to estradiol (E₂) and EGF, IGF‐I, or INS stimulated DNA labeling in the last 12 hr, especially when the cultures were treated with progression growth factors. bFGF pretreatment and subsequent treatment with E₂ for 36 hr stimulated DNA labeling. The 36‐hr E₂ treatment alone did not significantly decrease DNA labeling, but contemporary addition of E₂ with two or three growth factors stimulated DNA labeling remarkably. When E₂ was coadded with growth factors, a significantly increased DNA labeling was observed, demonstrating an astroglial synergistic mitogenic effect evoked by contemporary treatment with growth factors in the presence of estrogens. Cyclin D1 expression was markedly increased when astrocyte cultures were pretreated for 36 hr with E₂ and subsequently treated with two or three competence and progression growth factors. A highly significant increase of ERK1/2 expression was observed after all the treatments (EGF, bFGF, INS, IGF‐I alone or in combination with two or three growth factors). GFAP and vimentin expression was markedly increased when the cultures were treated with two or three growth factors. In conclusion, our data demonstrate estradiol–growth factor cross‐talk during astroglial cell proliferation and differentiation in culture. © 2015 Wiley Periodicals, Inc.     

Epidermal growth factor-induced cell proliferation in the adult rat striatum

Current strategies for repairing the adult CNS following injury include cell transplantation and/or the use of viral vectors to deliver therapeutic agents. Although promising, both techniques are limited in their usefulness due to the immunological response triggered in the brain as a result of the introduction of foreign antigens. An alternative method to repair the damaged CNS is to stimulate endogenous cells within the brain to divide thereby replacing cells lost to injury. Since it has been shown that growth factors such as epidermal growth factor (EGF) are potent mitogens to CNS cells in vitro, we sought to assess the mitogenic effect of an in vivo application of EGF to the adult mammalian brain. Accordingly, varying doses of human recombinant EGF were administered to the striatum of adult rats, followed 48 h later by intraperitoneal injections of 5-bromodeoxyuridine (BrdU), a marker for cell proliferation. Of four doses assessed, 0.05 ng of EGF induced the highest levels of cell proliferation. To determine the cellular identity of these proliferating cells, animals were injected with (3)H-thymidine 48 h following EGF administration to label dividing cells. Sections were subsequently immunostained for markers to astrocytes, microglia, oligodendrocytes, neural precursors, and mature neurons. Compared to controls, a significant proportion of the newly generated cells resulting from EGF administration were identified as immature and mature astrocytes. Collectively, these results provide valuable information for utilizing a growth factor administration approach to mobilize the proliferative response of endogenous cells to replace those lost to injury or disease.     

Receptor-mediated retrograde transport in CNS neurons after intraventricular administration of NGF and growth factors.

Radiolabel tracer techniques were used to follow the distribution of nerve growth factor (NGF) and other neuromodulatory factors after intraventricular injection. Autoradiography showed that shortly after intraventricular injection of radio-iodinated NGF (125I-NGF), substantial amounts of radioactivity had penetrated the ventricular wall surfaces; this binding was transient and nonspecific. The 125I-NGF was progressively cleared from the central nervous system (CNS), presumably via the flow of cerebrospinal fluid (CSF) into the blood. A relatively small proportion of the injected 125I-NGF was taken up by NGF receptor-positive neurons in the CNS. Retrograde accumulation of radiolabel was observed within the basal forebrain cholinergic neurons at 5 hours after intraventricular injection. Labeling intensity was maximal at 18 hours and much reduced by 30 hours. This labeling was blocked by co-injection of an excess of unlabeled NGF. Specific and saturable retrograde labeling was also observed within other NGF receptor-bearing neurons, including the prepositus hypoglossal nucleus and the raphe obscurus nucleus. When epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-beta 1), platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-I), or IGF-II was radiolabeled and injected intraventricularly, specific labeling of neurons was observed for 125I-IGF-II and 125I-LIF within separate subpopulations of the dorsal and medial raphe. No retrograde accumulation within neurons was observed for EGF, TGF-beta 1, PDGF-AA, PDGF-BB, or IGF-I. This study describes an in vivo method for identifying putative neuromodulatory factors and their responsive neurons.     

EGF and NGF injected into the brain of old mice enhance BDNF and ChAT in proliferating subventricular zone

The response of cells localized in the brain subventricular zone (SVZ) to growth factor stimulation has been largely described for development and adult life, whereas no information on their behavior during aging is available. To address the question of whether the cells in the SVZ of old mice respond to the intracerebroventricular administration of epidermal growth factor (EGF) and nerve growth factor (NGF), we studied the distribution of proliferating cells and the effects on ChAT and brain-derived neurotrophic factor (BDNF) synthesis in forebrain and SVZ. It was found that the conjoint administration of EGF + NGF produced a major increase in ChAT expression in both forebrain and SVZ. The ChAT mRNA levels and the number of ChAT positive cells localized in the ventricular border and in the parenchyma of SVZ area were also increased significantly in the mice receiving EGF + NGF. Enhanced numbers of SVZ cells expressing proliferative markers were also discovered in EGF + NGF treated mice and some of these cells expressed cholinergic markers, as demonstrated by double immunostaining. In addition, EGF and NGF treatments significantly upregulate BDNF protein and mRNA levels in this brain region. The present study demonstrates that cells localized in SVZ of aged mouse brain retain the capacity to respond to EGF and NGF and that after stimulation with these two growth factors, the synthesis of ChAT and BDNF also increases. The implication that cells of the SVZ remain a reservoir of cholinergic and BDNF-positive neurons in aged brain opens a new perspective for understanding the role of growth factors during neurodegenerative disorders associated with aging.     

Effects of pcDNA3-β-NGF Gene-modified BMSC on the Rat Model of Parkinson’s Disease

This study determined the effects of pcDNA3-beta-nerve growth factor (NGF) gene-modified bone marrow stromal cells (BMSC) on the rat model of Parkinson's disease (PD). The recombinant plasmid pcDNA3-beta-NGF was transfected into BMSC, and NGF expression and its biological activity in vitro were detected. BMSC modified by the NGF gene were then grafted into the corpus striatum of PD rats, and the rotation behavior was evaluated at 1, 2, 4, and 6 weeks post-transplantation. A significant improvement in rotation behavior was observed in PD rats subjected to cell transplantation, especially in PD rats receiving NGF-modified BMSC. The genetically modified BMSC survived and expressed beta-NGF but did not differentiate into tyrosine hydroxylase-positive cells in vivo. The present findings suggested that genetically modified BMSC could be effective for PD treatment, and the mechanisms might involve the neuroprotective effects of beta-NGF.