Injury-induced Regulation of Ciliary Neurotrophic Factor mRNA in the Adult Rat Brain

Ciliary neurotrophic factor (CNTF) is a pleiotropic molecule that acts as a neurotrophic factor for a wide range of embryonic neurons as well as a differentiation factor for sympathetic neuroblasts and O2A progenitor cells in culture. CNTF messenger RNA (mRNA) is present at very low levels in the normal adult rat central nervous system (CNS), but is dramatically up-regulated after an aspiration lesion of dorsal hippocampus and overlying cortex, in the area coincident with glial scar. The increased level of CNTF mRNA in lesioned hippocampus is maximal by 3 days and is sustained for up to 20 days, the longest time point examined. In contrast, mRNA levels for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were slightly decreased during the same period. In situ hybridization experiments revealed that cells expressing CNTF mRNA were concentrated at the margin of the wound, and also present within the gelfoam which filled the lesion cavity. This distribution of CNTF-expressing cells corresponded very closely to that of cells expressing high levels of glial fibrillary acidic protein mRNA at the wound site. Paralleling the observed increase in CNTF mRNA, increased levels of CNTF-like neurotrophic activity were apparent in soluble extracts of the lesioned tissues. This neurotrophic activity for ciliary ganglion neurons was completely blocked by the addition of neutralizing antiserum against CNTF. Basic fibroblast growth factor, which has been shown by others to increase after a similar lesion paradigm (Frautschy et al., Brain Res. , 553 , 291–299, 1991), does not contribute appreciably to this trophic activity. We conclude that CNTF is markedly increased as a function of injury to the CNS and that its expression is most likely restricted to reactive astrocytes in the glial scar.     

Neurotrophic properties of olfactory ensheathing glia

Olfactory ensheathing cells (OEC) constitute a specialized population of glia that accompany primary olfactory axons and have been reported to facilitate axonal regeneration after spinal cord injury in vivo. In the present report we describe OEC neurotrophic factor expression and neurotrophic properties of OECs in vitro. Investigation of the rat olfactory system during development and adulthood by radioactive in situ hybridization revealed positive labeling in the olfactory nerve layer for the neurotrophic molecules S-100beta, CNTF, BMP-7/OP-1, and artemin, as well as for the neurotrophic factor receptors RET and TrkC. Ribonuclease protection assay of cultured OEC revealed expression of NGF, BDNF, GDNF, and CNTF mRNA, while NT3 and NT4 mRNA were not detectable. In vitro bioassays of neurotrophic activity involved coculturing of adult OEC with embryonic chick ganglia and demonstrated increased neurite outgrowth from sympathetic, ciliary, and Remak's ganglia. However, when culturing the ganglia with OEC-conditioned medium, neurite outgrowth was not stimulated to any detectable extent. Our results suggest that the neurotrophic properties of OEC may involve secretion of neurotrophic molecules but that cellular interactions are crucial.     

Neurotrophic Factor Receptors and Their Signal Transduction Capabilities in Rat Astrocytes

Until recently, astrocytes were not considered as sites for neurotrophic factor action. We show here that, both in vivo and in vitro , astrocytes express receptors for two separate families of neurotrophic factors. In the intact adult rat CNS, astrocytes express the extracellular domain of the neurotrophin receptor TrkB and, in a more restricted population, the low-affinity nerve growth factor receptor p75^LNGFR. In the lesioned CNS, expression of the alpha component of the receptor for ciliary neurotrophic factor (CNTFRα) switches from a purely neuronal localization to cells in the glial scar at the edge of the wound. Using cultured hippocampal astrocytes as a model to address the functional status of these receptors, we have found only the truncated forms of TrkB and TrkC, which are incapable of signal transduction as measured by protein tyrosine phosphorylation or immediate early gene induction. In contrast, a fully functional CNTF receptor complex capable of signal transduction is present on cultured astrocytes. Thus, the neurotrophin receptors may act primarily to sequester or present the neurotrophins, whereas in the case of CNTF a functional response can be initiated within the astrocyte.     

Ciliary neurotrophic factor activates spinal cord astrocytes, stimulating their production and release of fibroblast growth factor-2, to increase motor neuron survival.

At focal CNS injury sites, several cytokines accumulate, including ciliary neurotrophic factor (CNTF) and interleukin-1beta (IL-1beta). Additionally, the CNTF alpha receptor is induced on astrocytes, establishing an autocrine/paracrine loop. How astrocyte function is altered as a result of CNTF stimulation remains incompletely characterized. Here, we demonstrate that direct injection of CNTF into the spinal cord increases GFAP expression and astroglial size and that primary cultures of spinal cord astrocytes treated with CNTF, IL-1beta, or leukemia inhibitory factor exhibit nuclear hypertrophy comparable to that observed in vivo. Using a coculture bioassay, we further demonstrate that CNTF treatment of astrocytes increases their ability to support ChAT(+) ventral spinal cord neurons (presumably motor neurons) more than twofold compared with untreated astrocytes. Also, the complexity of neurites was significantly increased in neurons cultured with CNTF-treated astrocytes compared with untreated astrocytes. RT-PCR analysis demonstrated that CNTF increased levels of FGF-2 and nerve growth factor (NGF) mRNA and that IL-1beta increased NGF and hepatocyte growth factor mRNA levels. Furthermore, both CNTF and IL-1beta stimulated the release of FGF-2 from cultured spinal cord astrocytes. These findings demonstrate that cytokine-activated astrocytes better support CNS neuron survival via the production of neurotrophic molecules. We also show that CNTF synergizes with FGF-2, but not epidermal growth factor, to promote DNA synthesis in spinal cord astrocyte cultures. The significance of these findings is discussed by presenting a new model depicting the sequential activation of astrocytes by cytokines and growth factors in the context of CNS injury and repair.     

Regulation of Ciliary Neurotrophic Factor in Cultured Rat Hippocampal Astrocytes

Ciliary neurotrophic factor (CNTF) is a pleiotrophic cytokine which is detectable only at very low levels in the intact adult rat CNS, but following an aspirative lesion to the dorsal hippocampus and overlying cortex, CNTF mRNA levels are dramatically up-regulated in reactive astrocytes. In cultured rat hippocampal astrocytes, CNTF mRNA levels are high, similar to the levels in reactive astrocytes in vivo , but are strongly suppressed after administration of isoproterenol and forskolin, which stimulate the production of intracellular cyclic AMP, induce marked morphological change in the astrocytes and up-regulate glial fibrillary acidic protein mRNA and nerve growth factor mRNA in these cells. Following a single administration of forskolin to cultured astrocytes, suppression of CNTF mRNA was sustained for up to 7 days. A similar down-regulation was observed with the endogenous adrenergic agonists noradrenaline and adrenaline as well as, to a lesser extent, dopamine and adenosine. Down-regulation of CNTF mRNA resulted in a gradual reduction in the level of CNTF protein within the astrocytes. A single addition of forskolin or isoproterenol resulted in a drop in CNTF protein levels to 29 and 52% of control levels respectively after 9 days in vitro , although the rate of turnover of CNTF remained the same. Down-regulation of CNTF mRNA in cultured hippocampal astrocytes by adenylyl cyclase activation was quite specific, as a wide range of growth factors, cytokines and neurotransmitters had little or no effect upon CNTF mRNA levels.     

Differential expression of mRNAs for the NGF family of neurotrophic factors in the adult rat central olfactory system.

The cellular localization of mRNAs for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3), in the rat central olfactory system was evaluated with in situ hybridization of 35S-labeled cRNA probes. In the main olfactory bulb, low levels of NGF and BDNF mRNA expression were detected. NGF mRNA was restricted to the glomerular region while BDNF mRNA was predominantly localized to the granule cell layer. No cellular hybridization to NT3 cRNA was seen. The accessory olfactory bulb did not express detectable levels of mRNA for any of the three related neurotrophic factors. Areas which receive olfactory bulb afferents expressed comparatively high levels of both NGF and BDNF mRNA. Cell labeling with cRNAs for NGF and BDNF occurred throughout the cellular layers of the anterior olfactory nucleus and in layers 2 and 3 of rostral piriform cortex. BDNF mRNA expression in these areas appeared more robust than that of NGF mRNA, while NT3 mRNA was not detectable. In contrast, tenia tecta exhibited dense labeling with the cRNAs for all three neurotrophic factors. The localization of NGF mRNA to primary target neurons of the olfactory nerve in the periglomerular region of the main olfactory bulb suggests that bulb cells may influence the ingrowth and continual turnover of olfactory sensory afferents. However, as there is a strong correlation between the distribution of neurotrophic factor mRNAs within rostral olfactory structures and the distribution of centrifugal cholinergic afferents, it is more likely that bulb-derived NGF, and possibly BDNF, act on the cholinergic neurons of the basal forebrain.     

Traumatized Rat Striatum Produces Neurite-Promoting and Neurotrophic Activitiesin Vitro

We have previously reported that ciliary neurotrophic factor (CNTF) mRNA is upregulated in the rat striatum following trauma and that its peak is coincident with a peak in the number of GFAP-positive astrocytes. CNTF, or other neurotrophic factors present in the traumatized striatum, may be involved in the dopaminergic fiber sprouting seen following cavitation or graft implantation in animal models of Parkinson's disease. This study was undertaken in order to further characterize the neurotrophic activity present following trauma through the use of bioassays. Adult rats underwent stereotaxic biopsy of the right striatum, and gelatin sponge [gelfoam (GF)] was placed in the resultant cavity. GF was collected from 1 to 30 days following trauma and homogenized. GF extracts (with equal protein concentrations) were assayed using dorsal root ganglion (DRG) explants, dissociated ciliary ganglia (CG), and human dopaminergic neuroblastoma cell (SH-SY5Y) cultures. The GF extracts had significant neurite-promoting activity (NPA) for DRG, CG, and SH-SY5Y cells, with the maximum effect seen 7 days after trauma. NPA was not blocked by anti-nerve growth factor (NGF) Ab, but anti-brain-derived neurotrophic factor (BDNF) Ab significantly blocked the activity for DRG. The GF extracts protected the SH-SY5Y cells from the neurotoxins 6-OHDA and MPP⁺, as did NGF and BDNF. This neuroprotective effect of GF was not blocked by anti-NGF Ab. This study suggests that the neurotrophic activity in GF extracts has CNTF-like and BDNF-like components as well as another, undefined component.     

Complexity in the modulation of neurotrophic factor mRNA expression by early visual experience

The expression of mRNA for brain-derived neurotrophic factor (BDNF) is regulated by early visual experience. In this study, we sought to determine whether other neurotrophic factor mRNAs are similarly regulated. We reared pigmented rats from birth to postnatal day 21 in a normal light cycle, constant light (LR) or constant darkness (DR). In the retina, superior colliculus (SC), primary visual cortex (V1), hippocampus (HIPP) and cerebellum (CBL), using a ribonuclease protection assay (RPA), we examined expression of the mRNAs for nerve growth factor (NGF), BDNF, NT3, NT4, ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF). LR or DR alter the expression of the mRNAs for NGF, BDNF and NT3 and CNTF within the visual system. LR also upregulated BDNF mRNA expression within the cerebellum. In all of the structures examined, NT4 mRNA expression was unaltered by LR or DR and GDNF mRNA was undetectable. Notably, the same rearing condition could induce changes of opposite sign in the mRNA for a single factor in different structures or for different factors in the same structure. Thus, during developmental stages when sensory experience and neuroelectric activity are important in the shaping of visual circuitry, vision regulates the expression of multiple neurotrophic factor mRNAs and each mRNA has a unique profile with respect to the locus and sign of activity-induced changes.     

Cerebrolysin modulates pronerve growth factor/nerve growth factor ratio and ameliorates the cholinergic deficit in a transgenic model of Alzheimer's disease

Alzheimer's disease (AD) is characterized by degeneration of neocortex, limbic system, and basal forebrain, accompanied by accumulation of amyloid‐β and tangle formation. Cerebrolysin (CBL), a peptide mixture with neurotrophic‐like effects, is reported to improve cognition and activities of daily living in patients with AD. Likewise, CBL reduces synaptic and behavioral deficits in transgenic (tg) mice overexpressing the human amyloid precursor protein (hAPP). The neuroprotective effects of CBL may involve multiple mechanisms, including signaling regulation, control of APP metabolism, and expression of neurotrophic factors. We investigate the effects of CBL in the hAPP tg model of AD on levels of neurotrophic factors, including pro‐nerve growth factor (NGF), NGF, brain‐derived neurotrophic factor (BDNF), neurotropin (NT)‐3, NT4, and ciliary neurotrophic factor (CNTF). Immunoblot analysis demonstrated that levels of pro‐NGF were increased in saline‐treated hAPP tg mice. In contrast, CBL‐treated hAPP tg mice showed levels of pro‐NGF comparable to control and increased levels of mature NGF. Consistently with these results, immunohistochemical analysis demonstrated increased NGF immunoreactivity in the hippocampus of CBL‐treated hAPP tg mice. Protein levels of other neurotrophic factors, including BDNF, NT3, NT4, and CNTF, were unchanged. mRNA levels of NGF and other neurotrophins were also unchanged. Analysis of neurotrophin receptors showed preservation of the levels of TrKA and p75^NTR immunoreactivity per cell in the nucleus basalis. Cholinergic cells in the nucleus basalis were reduced in the saline‐treated hAPP tg mice, and treatment with CBL reduced these cholinergic deficits. These results suggest that the neurotrophic effects of CBL might involve modulation of the pro‐NGF/NGF balance and a concomitant protection of cholinergic neurons. © 2012 Wiley Periodicals, Inc.     

睫状神经营养因子对体外培养星形胶质细胞的激活作用(英文)

目的观察睫状神经营养因子(ciliary neurotrophic factor, CNTF)对体外培养星形胶质细胞的细胞激活作用。方法分别给予不同浓度(0、2、20、200 ng/ml)的CNTF孵育有血清培养和无血清培养的星形胶质细胞,采用免疫细胞化学技术及流式细胞术,观察星形胶质细胞形态及细胞周期的变化。结果有血清培养和无血清培养时CNTF均使星形胶质细胞GFAP表达增强,胞核增大。有血清培养时CNTF还可以促进星形胶质细胞进入细胞周期进行增殖;无血清培养时CNTF无此效应。结论无血清培养时CNTF可以刺激星形胶质细胞进入活化状态,但不刺激其增殖;有血清培养时CNTF可以协助血清中的丝裂原引起星形胶质细胞增殖。     

Expression of mRNA for brain-derived neurotrophic factor in the dorsal root ganglion following peripheral inflammation

It is well known that the nerve growth factor (NGF) may serve as a link between inflammation and hyperalgesia. Recent experiments showed that systemic injection of NGF dramatically stimulated the expression of brain-derived neurotrophic factor (BDNF) mRNA in the dorsal root ganglion (DRG). In the present study, we evaluated the change of BDNF mRNA in the DRG following peripheral inflammation and also observed colocalization of BDNF and trkA mRNAs by means of in situ hybridization histochemistry in rats. Peripheral tissue inflammation produced by an intraplantar injection of Freund's adjuvant into the paws significantly increased BDNF mRNA levels in the DRG and many neurons expressing trkA mRNA showed increased expession of BDNF mRNA. Intraplantar injection of antibody to NGF together with Freund's adjuvant prevented the increase in BDNF mRNA. These findings suggest that peripheral inflammation induces an increased expression of BDNF mRNA which is mediated by NGF in DRG.     

Nerve Growth Factor Regulates Expression of the Nerve Growth Factor Receptor Gene in Adult Sensory Neurons

Sensory neurons of the adult rat dorsal root ganglion (DRG) can be maintained in culture in the absence of nerve growth factor (NGF). We have thus used dissociated cultures of these neurons to study effects of NGF on the regulation of expression of mRNA encoding the nerve growth factor receptor (NGF-R). In the absence of NGF, levels of NGF-R mRNA remained constant for 7 days in cultures of adult rat DRG neurons. In the presence of NGF, NGF-R mRNA levels rose two - three-fold after 2 days, reaching plateau levels (five - six-fold elevation) after 5 days. This NGF-induced up-regulation could be demonstrated even after prior NGF-deprivation for 3 - 4 days. NGF had no effect upon NGF-R mRNA levels in DRG non-neuronal cells. Epidermal growth factor (EGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF) were without effect on NGF-R mRNA levels, but 8-bromo-cAMP decreased NGF-R mRNA levels by 65% after 2 days. NGF also induced a rapid (30 min) rise in expression of c-fos in DRG neurons, but not in non-neuronal cells. Our results suggest that endogenous levels of NGF may regulate the expression of NGF-R in vivo.     

睫状神经营养因子对体外培养星形胶质细胞的激活作用

目的观察睫状神经营养因子（ciliary neurotrophic factor,CNTF）对体外培养星形胶质细胞的细胞激活作用。方法分别给予不同浓度（0、2、20、200ng／ml）的CNTF孵育有血清培养和无血清培养的星形胶质细胞,采用免疫细胞化学技术及流式细胞术,观察星形胶质细胞形态及细胞周期的变化。结果有血清培养和无血清培养时CNTF均使星形胶质细胞GFAP表达增强,胞核增大。有血清培养时CNTF还可以促进星形胶质细胞进入细胞周期进行增殖：无血清培养时CNTF无此效应。结论无血清培养时CNTF可以刺激星形胶质细胞进入活化状态,但不刺激其增殖：有血清培养时CNTF可以协助血清中的丝裂原引起星形胶质细胞增殖。     

Pathology-related differential expression regulation of NGF, GDNF, CNTF, and IL-6 mRNAs in human vasculitic neuropathy

The mRNA levels of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and interleukin-6 (IL-6) were examined in sural nerves of 22 patients with acute necrotizing vasculitic neuropathies. NGF, GDNF, and IL-6 mRNAs were upregulated and CNTF mRNA was downregulated in the lesioned nerves, but their up- and down-regulation levels were not correlated with each other, showing that these mRNAs were independently expressed. The expression of NGF and CNTF mRNAs was clearly correlated with the degree of infiltrated macrophages and T cells, and myelinated fiber density, respectively. These findings indicate that these neurotrophic factors are differentially expressed temporally and spatially in the vasculitic nerve lesion by an underlying pathology-related process.     

The Neurotrophins BDNF, NT-3 and NT-4/5, But Not NGF, Up-regulate the Cholinergic Phenotype of Developing Motor Neurons

Although developing motor neurons express low-affinity nerve growth factor (NGF) receptors, there is no known biological effect of NGF on developing or adult motor neurons. In this study, we found that, unlike NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) stimulated cholinergic phenotype by increasing choline acetyltransferase (CAT) activity in cultures enriched with embryonic rat motor neurons. Ciliary neurotrophic factor (CNTF) also stimulated CAT activity. The effects of BDNF and NT-4/5 on CAT activity appeared to be synergistic with that of CNTF. Cotreatment with BDNF and NT-3 resulted in an additive effect, suggesting that signal transduction was mediated through different high-affinity receptors tyrosine kinases B and C (Trk B and Trk C). However, cotreatment with BDNF and NT-4/5 did not result in an increase in CAT activity greater than that of either BDNF or NT-4/5 alone, suggesting that their effects were mediated via the same receptor Trk B. Supporting our findings that spinal cholinergic neurons are responsive to trophic actions of members of the neurotrophin family, motor neuron-enriched cultures were found to express mRNA for Trk B and Trk C, which have been identified as high-affinity receptors for BDNF and NT-4/5, and NT-3, respectively.     

Skeletal muscle extract and nerve growth factor have developmentally regulated survival promoting effects on distinct populations of mammalian sensory neurons

Neurotrophic factors appear to be relevant to the therapy of degenerative diseases as well as neural regeneration. In this respect, we have investigated the neurotrophic effects of skeletal muscle extract on DRG neuron survival by examining the survival and neurite outgrowth promoting activity of factor(s) present in skeletal muscle extracts (SME) on dissociated cultures of embryonic or early postnatal mouse dorsal root ganglion (DRG) sensory neurons. The numbers of surviving neurons resulting from SME addition increased continuously from embryonic day 13 (15%) to birth (55%), then decreased up to 7 days after hatching (0%). Preliminary characterization of the factor(s) present in SME suggests that the active molecule is a protein different from the known neurotrophic factors NGF, BDNF, NT3, CNTF, and bFGF, and that its neurotrophic effect is not mediated by direct interaction with the substratum. © 1993 John Wiley & Sons, Inc.     

In vivo survival requirement of a subset of nodose ganglion neurons for nerve growth factor

The sensory neurons of the nodose ganglion are the classic example of a population of peripheral nervous system neurons that do not require nerve growth factor (NGF) for survival during development but are dependent on other neurotrophins. We have re-examined this assertion by studying the development of the nodose ganglion of mice that have a null mutation in the NGF gene. Compared with wild-type embryos, the number of neurons undergoing apoptosis was elevated in NGF -/- mice, resulting in a significant reduction in the total number of neurons in the ganglion by the end of embryonic development. TrkA, the NGF receptor tyrosine kinase, was expressed in the nodose ganglion throughout development and there was a marked decrease in TrkA mRNA expression in the nodose ganglion of NGF -/- embryos. Although the in vitro survival of the majority of nodose neurons was promoted by brain-derived neurotrophic factor (BDNF), a minor proportion was supported by NGF in cultures established over a range of embryonic stages. These results clearly demonstrate that a subset of nodose ganglion neurons depends on NGF for survival during development. The finding that the expression of tyrosine hydroxylase (TH) mRNA was unaffected in the nodose ganglia of NGF-deficient embryos indicates that this NGF-dependent subset is distinct from the subset of catacholaminergic neurons in the nodose ganglion.     

Role of Bcl-2 in the Brain-derived Neurotrophic Factor Survival Response

Developing neurons die if they fail to obtain an adequate supply of neurotrophins from their targets but how neurotrophins suppress cell death is not known. Although over-expression of exogenous Bcl-2 can prevent the death of cultured neurons deprived of members of the nerve growth factor family of neurotrophins it is not known if this effect is physiologically relevant. To determine if Bcl-2 participates in the neurotrophin survival response we used antisense bcl-2 RNA to inhibit endogenous Bcl-2 expression. Here we show that brain-derived neurotrophic factor (BDNF)-dependent neurons are killed by antisense bcl-2 RNA in the presence of BDNF. However, when these neurons were supported with ciliary neurotrophic factor (CNTF) their survival was not affected by antisense bcl-2 RNA. Likewise, the survival of CNTF-dependent ciliary neurons was not affected by antisense bcl-2 RNA. Our findings suggest that Bcl-2 is required for the BDNF survival response and that alternative, Bcl-2-independent survival mechanisms operate in sensory and parasympathetic neurons exposed to CNTF.     

Survival of purified embryonic chick retinal ganglion cells in the presence of neurotrophic factors

In a search for neurotrophic factors (NTFs) regulating retinal ganglion cell (RGC) death in the chick embryo we have used purified and cultured RGCs. Purification of RGCs from embryonic day 10 was achieved by employing the "panning" method (Silverstein and Chun: Soc Neurosci Abstr 13:1054, 1987). The obtained neuron population consisted of 97% RGCs as demonstrated by retrograde labeling with a fluorescence dye. RGCs were cultured at low density in a chemically defined medium and short-term survival (24 hr) was determined. In the absence of NTFs, less than 3% of the RGCs survived. In the presence of various crude or purified NTFs (eye, brain, and tectum extracts; glial-conditioned medium; ciliary neurotrophic factor [CNTF]; nerve growth factor [NGF]) 31% to 52% of the RGCs were maintained. The effects of NGF and CNTF were not additive. Neither acidic nor basic fibroblast growth factor was able to maintain RGCs in culture. Our results, obtained with a culture system which allowed the analysis of direct trophic actions, suggest that NGF and CNTF may be NTFs for overlapping subpopulations of chick RGCs.