神经生长因子和脑源性神经营养因子在脑胶质瘤内的表达

目的研究NGF(NeverGrowth Factor,神经生长因子)、BDNF(BrainDerivedNeurotrophicFactor,脑源性神经营养因子)在脑胶质瘤内的表达,探讨它们与肿瘤病理分级、实体肿瘤部位的关系,以及它们在肿瘤发生过程中的作用。方法通过免疫组织化学SP染色方法检测70例胶质瘤和5例正常脑组织中NGF和BDNF的表达。结果NGF和BDNF阳性细胞表达率明显高于正常脑组织内的表达,有统计学意义(P<0.05);在不同病理级别胶质瘤之间阳性细胞表达率也有差异,分别是:I级16.56%,11.24%;II级32.45%,18.23%;III级40.91%,21.44%;IV级24.71%,15.39%,正常脑组织内的表达率为7.06%,5.98%,且有统计学意义(P<0.05)。NGF和BDNF阳性细胞表达率在不同部位胶质瘤内各不相同,分别为:颞叶42.57%,24.19%;顶叶35.62%,20.09%;小脑28.67%,16.17%;额叶21.45%,12.42%,差异有显著性(P<0.05)。结论NGF和BDNF在脑胶质瘤内高表达;NGF和BDNF阳性细胞表达与胶质瘤的病理级别、肿瘤部位有关;NGF和BDNF可能参与了胶质瘤的发生。     

Anterograde axonal transport of BDNF and NT-3 by retinal ganglion cells: roles of neurotrophin receptors

Retinal ganglion cells (RGCs) transport exogenous neurotrophins anterogradely to the midbrain tectum/superior colliculus with significant downstream effects. We determined contributions of neurotrophin receptors for anterograde transport of intraocularly injected radiolabeled neurotrophins. In adult rodents, anterograde transport of brain-derived neurotrophic factor (BDNF) was receptor-mediated, and transport of exogenous BDNF and neurotrophin-3 (NT-3) was more efficient, per RGC, in rodents than chicks. RT-PCR and Western blot analysis of purified murine RGCs showed that adult RGCs express the p75 receptor. Anterograde transport of BDNF or NT-3 was not diminished in p75 knock-out mice (with unaltered final numbers of RGCs), but BDNF transport was substantially reduced by co-injected trkB antibodies. In chick embryos, however, p75 antisense or co-injected p75 antibodies significantly attenuated anterograde transport of NT-3 by RGCs. Thus, neither BDNF nor NT-3 utilizes p75 for anterograde transport in adult rodent RGCs, while anterograde NT-3 transport requires the p75 receptor in embryonic chicken RGCs.     

Synergistic Trophic Actions on Rat Basal Forebrain Neurons Revealed by a Synthetic NGF/BDNF Chimaeric Molecule

Basal forebrain cholinergic neurons, which degenerate in Alzheimer's disease, respond to multiple trophic factors, including the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). This dual responsiveness prompted us to investigate the effects of a synthetic chimaeric molecule, containing the active domains of both NGF and BDNF. The NGF/BDNF chimaeric factor exhibited synergistic actions, and was 100-fold more potent than wild-type BDNF in enhancing survival of cultured dissociated basal forebrain cholinergic neurons. This effect was apparently due to true BDNF/NGF synergy, since addition of the two wild-type trophins simultaneously reproduced the effect of the chimaera. Synergy was selective for neurons which respond to both factors; substantia nigra dopaminergic neurons, which respond to BDNF but not NGF, exhibited no potentiation. The chimaeric factor thus revealed a synergy that may normally occur in the brain, and constitutes a potentially novel therapeutic agent with greater potency than naturally occurring individual trophins.     

Regulation of Neuropeptides in Adult Rat Forebrain by the Neurotrophins BDNF and NGF

The expression of neuropeptides and neurotrophic factors is altered in the hippocampus after seizure induction in rats. Because the increase in brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) mRNAs precede changes in neuropeptide expression after seizure, it is possible that BDNF and NGF mediate subsequent alterations in peptide expression. To test this hypothesis directly, BDNF or NGF was infused into the hippocampus and cortex of adult rats. To ascertain the regional specificity of any observed effects of neurotrophin administration on neuropeptide expression, infusions into the striatum were also studied. To control for specificity, vehicle was also infused into the same sites. Peptide and mRNA alterations were assessed by Northern analysis, immunohistochemistry and radioimmunoassay. BDNF produced elevations of peptide and mRNA for neuropeptide Y and cholecystokinin in hippocampus and cortex, and somatostatin in cortex. BDNF increased mRNAs for neuropeptide Y, cholecystokinin, substance P and dynorphin in striatum. In contrast, BDNF decreased dynorphin peptide and mRNA in hippocampus. NGF's effects were limited to small mRNA increases, without corresponding changes in peptide levels, for neuropeptide Y in hippocampus and striatum, substance P in cortex and cholecystokinin in striatum. The distinct and limited effects of NGF infusion on neuropeptide expression demonstrate that BDNF's effects are not non-specific results of protein infusion into the brain. These findings indicate that BDNF may play a regionally specific role in modulating neuropeptide expression in the normal brain as well as in various pathophysiological states.     

BDNF and NGF treatment in lesioned rats: effects on cholinergic function and weight gain.

Effects of chronic intraventricular administration of recombinant human brain-derived neurotrophic factor (rhBDNF) or recombinant human nerve growth factor (rhNGF) on presynaptic hippocampal cholinergic function in adult rats with partial fimbrial transections were measured. Partial fimbrial transections reduced synaptosomal high affinity choline uptake, choline acetyltransferase activity, and [3H] acetylcholine synthesis by approximately 50-75%. Chronic treatment with rhBDNF failed to attenuate these lesion-induced decreases. In contrast, chronic rhNGF treatment increased all three parameters by 50-90% compared to lesioned control values. Chronic treatment with rhBDNF or rhNGF attenuated weight gain of the animals. The findings failed to provide evidence for a prominent role of BDNF in the function of adult cholinergic neurons, however, they suggest an action on central neurons involved in the regulation of food intake.     

Dynamic patterns of BDNF expression in injured sensory neurons: differential modulation by NGF and NT-3

It has been suggested that altered retrograde neurotrophin support contributes to the phenotypic switch observed in BDNF expression in injured sensory neurons. Thus, modulatory influences of NGF and NT-3 on BDNF expression in injured adult rat DRG neurons were examined using in situ hybridization and immunohistochemical approaches. Quantitative analysis reveals a biphasic response to sciatic nerve injury, whereby in the first day following injury, BDNF expression is up-regulated in approximately 83% of injured neurons including all small neurons, and a larger pool of trkB expressing neurons than in intact. By 1 week and up to 3 weeks later expression is still seen in approximately 66% of injured neurons, but the characteristic phenotypic switch in the subpopulations expressing BDNF occurs, whereby expression in the trkA population is reduced and expression in trkB- and in trkC-positive neurons is elevated. NGF infusion results in elevated levels of BDNF expression in both intact and injured trkA-positive neurons, accompanied by reduced trkB expression. NT-3 acts in an opposite fashion effecting a down-regulation in BDNF expression in intact neurons and preventing/reducing the injury-associated increases in BDNF expression in both trkC- and nontrkC-expressing subpopulations of injured neurons. These effects suggest NGF can regulate BDNF expression in trkA-expressing neurons regardless of the axonal state and that elevated levels of BDNF may contribute to the down-regulation in trkB expression associated with these states. Furthermore, the findings demonstrate that NT-3 can act in an antagonistic fashion to NGF in the regulation of BDNF expression in intact neurons, and mitigate BDNF's expression in injured neurons.     

Improvement of the specificity of dipetarudin by site directed mutagenesis

Protease specificity is crucial to the design of thrombin inhibitors as inhibition of other physiologically relevant serine-proteases can compromise their clinical use. Dipetarudin, a potent thrombin inhibitor, also inhibits trypsin and plasmin. Due to the specificity of an inhibitor being influenced by the amino acid residue at the P1 position, we replaced the Arg10 at P1 position of dipetarudin by a histidine, which is the P1 residue of rhodniin, a very specific thrombin inhibitor. The amino acid replacement was carried out by site directed mutagenesis. The mutant, dipetarudinR10H, showed a loss of plasmin and trypsin inhibitory activities present in its wild-type counterpart and a 3-fold higher dissociation constant for thrombin than dipetarudin. However, compared to dipetarudin and r-hirudin, dipetarudinR10H showed similar activity in coagulation screening assays such as activated partial thromboplastin time (aPTT), prothrombin time (PT), ecarin clotting time (ECT) and ecarin chromogenic assay (ECA).     

神经营养因子NGF、BDNF对AD模型鼠海马移植的影响

目的:研究神经营养因子NGF、BDNF对AD模型鼠海马移植后行为和形态学变化。方法:24.只AD模型鼠随机分成4组:单纯胚基底前脑细胞悬液移植组(ST组)、含NGF或BDNF胚基底前脑细胞悬液移植组(NGF组)、(BD-NF组)和模型对照组(M组),移植后3月进行行为测试并比较移植区AchE细胞数和纤维密度,运用方差分析和SNK检验进行组间比较。结果:行为测试移植3组明显优于模型M组,含因子组又较ST组效果好(P<0.01),两因子组间差异无显著性(P>0.05);因子组存活细胞数均高于ST组,NGF组细胞数多于BDNF组(P<0.05),纤维密度两组相似(P>0.05)。结论:海马内存活胆碱能神经元能代偿受损胆碱能神经元的功能,改善动物的学习记忆功能;NGF、BDNF均能促进胆碱能神经元存活,增加AchE细胞数目和突起,但BDNF促进神经元突起延伸作用较好,而NGF则对神经元保护作用较强。     

Thyroid hormone regulation of NGF, NT-3 and BDNF RNA in the adult rat brain.

The effects of peripherally administered thyroid hormone (TH; 500 micrograms/kg; i.p.; q.d.) on the relative abundances of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) RNA were determined by rtPCR in the cortex and hippocampus of young adult rats. Corresponding changes in choline acetyltransferase (ChAT) activity were measured since NGF and BDNF have been shown to enhance the expression of this marker enzyme of central cholinergic pathways. Abundance levels of NGF and NT-3, relative to cyclophilin (cycl), were increased significantly (+50%, P < 0.05) in the hippocampus following TH treatment. Despite enhanced abundance of NGF in the hippocampus, ChAT activity was unchanged, whereas ChAT activity was modestly increased by 28% in the cortex without corresponding changes in NGF, NT-3 or BDNF. These results demonstrate that TH administration is capable of inducing the accumulation of NT-3, in addition to NGF but that the induction levels of RNA cannot be directly correlated with responsivity of the cholinergic system as measured by ChAT activity.     

Measurement of neurotrophin 4/5 in rat tissues by a sensitive immunoassay.

Neurotrophin 4/5 (NT4/5) is a member of the neurotrophin family known to exert survival and other effects on a variety of neurons including those within the motor, sensory and central populations. Although mRNA(NT4/5) has been found in various effector tissues of the rat and human, the concentration of NT4/5 protein in tissues has not been reported previously due to lack of suitable methodology. We present here a quantitative two-site enzyme-linked immunosorbent assay for the estimation of NT4/5 in pre- and postnatal rat tissues. The assay was performed using a combination of polyclonal and monoclonal antibodies to recombinant human NT4/5. Tissue samples were extracted at neutral pH. Results show that the assay is highly specific for NT4/5 with a sensitivity of 1 pg/ml, and reproducible with intra- and inter-assay variation coefficients of 3.0 and 6.3%, respectively. NT4/5 was found in most embryonic tissues examined at gestation day 17 and 21, but was rarely detectable in postnatal tissues, with the notable exception of the testis. The availability of an immunoassay for the estimation of NT4/5 protein in rat tissues should contribute to the understanding of the physiology of this little understood neurotrophic factor.     

Human monocyte/macrophages activate by exposure to LPS overexpress NGF and NGF receptors

Monocyte/macrophages (M/M) represent the main cellular component of the immune system involved in the inflammatory response. In the present study we investigate whether NGF is produced by M/M and is involved in this event. The results show that unstimulated human M/M produce NGF and its synthesis is stimulated by LPS. The increase of NGF is associated with enhanced expression of high affinity NGF receptor on M/M and with no changes of low affinity NGF receptors (p75). The neutralization of endogenous NGF by NGF antibody in LPS-activated M/M, leads to overexpression of p75 receptor causing apoptosis. These findings provide new insight in the mechanisms governing monocyte survival in the inflamed tissue, representing a crucial aspect of host defence and maintenance of homeostasis.     

Time-dependence and cell-type specificity of synergistic neurotrophin actions on spiral ganglion neurons

The neurotrophins brain-derived neurotrophin (BDNF) and neurotrophin-3 (NT-3) synergistically enhance survival of spiral ganglion neurons such that simultaneous exposure to both compounds produces a larger response than would be expected from their individual effects. To elucidate the functional role of this neurotrophin interaction, we examined its temporal and cell-type specificity in vitro for both mouse and gerbil spiral ganglion neurons. Synergistic effects were transient; they were maximal within the first two postnatal days and declined during the first postnatal week. Both neurotrophins were, however, still efficacious at increasing cell survival. After postnatal day 10, the effects of coexposure to BDNF and NT-3 were additive rather than synergistic. Synergism declined more rapidly in mouse than gerbil neurons, reflecting the difference in cochlear development for each species. Only neurons without peripherin epitopes, putative type I neurons, showed synergistic survival effects; survival of peripherin-expressing neurons was purely additive. Therefore, during a restricted time period, identical neurotrophin stimuli are capable of preferentially enhancing survival of one class of neurons that compose approximately 95% of the adult spiral ganglion. J. Comp. Neurol. 402:129–139, 1998. © 1998 Wiley-Liss, Inc.     

Direct and indirect evidences of BDNF and NGF as key modulators in depression: role of antidepressants treatment

Purpose: Depression is one of the most prevalent, recurrent and life-threatening mental illnesses. However, the precise mechanism underlying the disorder is not yet clearly understood. It is therefore, essential to identify the novel biomarkers which may help in the development of effective treatment.

Methods: In this milieu, the profile of the brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were considered as biomarkers in the light of pathophysiology of depression and its treatment.

Results: Previously, we have reported that BDNF level in the postmortem brain of suicide victims was significantly lower than those of normal controls. We also found decreased BDNF levels in the specific brain regions of the learned helplessness model of depression in rat, and was found to increase normal level following chronic fluoxetine hydrochloride treatment. NGF is another important member of neurotrophin, which is dysregulated in the pathophysiology of depression in some models of peripheral nerve damage and stress. The results shown evidences of the effect of antidepressants on modulating depression via the NGF in preclinical and clinical models of depression, but conflicted, therefore make it currently difficult to affirm the therapeutic role of antidepressants.

Conclusions: Here, we review some of the preclinical and clinical studies aimed at disclosing the role of BDNF and NGF mediated pathophysiological mechanisms of depression and the new therapeutic approaches targeting those key molecules. In addition, an important link between BDNF, NGF and depression has been discussed in the light of current existing knowledge.     

Embryonic Precursor Cells That Express Trk Receptors: Induction of Different Cell Fates by NGF, BDNF, NT-3, and CNTF

Epidermal growth factor (EGF)-treated neurosphere cultures from embryonal striatum contain multipotential cells capable of neuronal, astrocytic, and oligodendroglial differentiation. In this study, we tested whether these neural precursor cells differentiate in the presence of neurotrophic factors. We first assayed neurosphere cells for expression of neurotrophin receptors. TrkA, TrkB, TrkC, and gp75 were detected by immunofluorescence microscopy in 60–80% of cells. In addition, the ciliary neurotrophic factor receptor α was expressed in 50–60% of cells. In the presence of the mitogen, EGF, treatment of stem cells with neurotrophic factors had no apparent effect. Removal of EGF from cells resulted in cessation of cell proliferation and pronounced astrocytic (glial fibrillary acidic protein⁺) differentiation. Neuronal (neurofilament⁺) and oligodendroglial (galactocerebroside⁺) cells appeared in cultures treated with neurotrophic factors. Nerve growth factor (NGF) resulted in bipolar neuronal cells, and brain-derived neurotrophic factor led to multipolar neuronal cells. Treatment with neurotrophin-3 or ciliary neurotrophic factor resulted in bipolar neuronal cells and oligodendrocytes. Neuronal differentiation in the presence of NGF was enhanced by extracellular matrix, and the resulting neuronal cells expressed choline acetyltransferase and, to a lesser degree, tyrosine hydroxylase. These studies demonstrate that neurotrophic factors influence the fates of these multipotential precursor cells. Indeed, the true utility of multipotential precursor cells is the production of different types of cells in different situations. Local cues, such as neurotrophic factors and extracellular matrix, may regulate production of different types of neural cells during development or in response to other stimuli, such as injury.     

The effects of intrahippocampal BDNF and NGF on spatial learning in aged long evans rats

Spatial learning rate was compared in cognitively impaired aged rats infused with either brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF). BDNF or NGF was infused into the dorsal hippocampus/third ventricle while animals were being trained on the Morris water maze. Training continued until all rats met a spatial learning criterion. Seven weeks later, they were tested for retention of the task, and sacrificed for assessment of hippocampal high-affinity choline uptake (HACU) or hypothalamic biogenic amine levels. NGF, but not BDNF, improved spatial learning rate in aged rats and increased hippocampal choline uptake weeks after withdrawal of NGF. Although BDNF did not improve, spatial learning, it did induce a partial, long-term normalization of the elevated hypothalamic 5-HT levels observed in our aged rats. These data suggest that (1) intrahippocampal/intraventricular infusion of NGF can improve the learning rate of aged, spatial learning-impaired rats, and that this improvement in acquisition could be associated with increased hippocampal cholinergic activity, and (2) that the BDNF-induced normalization of hypothalamic 5-HT levels in aged rats was not sufficient to improve learning rate in aged, spatial learning-impaired rats.     

嗅鞘细胞及其表达的NGF和BDNF对神经干细胞增殖的影响

目的 研究嗅鞘细胞及其表达的神经生长因子(NGF)和脑源性神经营养因子(BDNF)对神经干细胞增殖的影响.方法 采用共培养液培养以及NGF或BDNF抗体封闭的方法,观察嗅鞘细胞对神经干细胞增殖的影响.免疫组化和RT.PCR半定量分析嗅鞘细胞表达的细胞因子及其受体的情况.结果 共培养液培养4d后,神经干细胞数量明显增多(P<0.05).免疫组化和RT-PCR半定量分析结果显示,嗅鞘细胞表达多种细胞因子及受体,共培养液培养2d后嗅鞘细胞表达NGF和BDNF mRNA的相对值明显高于对照组(P<0.05).抗体封闭培养液中的NGF或BDNF后,没有影响神经干细胞的增殖.结论 嗅鞘细胞表达大量细胞因子作用于神经干细胞,促进其增殖.在本研究条件下,神经干细胞的增殖可能与嗅鞘细胞表达的碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)有关,而与NGF和BDNF无关.