Overexpression of ciliary neurotrophic factor in vivo rescues chick ciliary ganglion neurons from cell death

Ciliary ganglion (CG) neurons undergo target-dependent cell death during embryonic development. Although ciliary neurotrophic factor (CNTF) was identified in vitro by its ability to support the survival of chick CG neurons, its function as a target-derived neurotrophic factor has been questioned by those working on mammalian-derived forms of CNTF. We have purified and cloned a chicken CNTF [chCNTF; formerly growth-promoting activity (GPA)] that is expressed in CG targets during the period of cell death and is secreted by cells transfected with chCNTF. In the present study we used a retroviral vector, RCASBP(A), to overexpress chCNTF in CG target tissues. Elevation of chCNTF biological activity three- to fourfold in the embryonic eye rescued an average of 31% of the neurons that would have normally died in vivo. In some individuals, nearly all of the neurons were rescued. ChCNTF had no effect on the number of neurons observed prior to cell death, nor were there any deleterious effects of either viral infection or overexpression of CNTF. These results show that chCNTF is able to function in vivo as a trophic factor for CG neurons, and suggest that limited availability of trophic support is one of the factors regulating CG neuron survival during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 283–293, 1998     

Expression,localization, and function of transforming growth factor-βs in embryonic chick spinal cord,hindbrain, and dorsal root ganglia

We have studied the localizations of transforming growth factor-beta (TGF-β) 2 and 3 immunohistochemically using isoform-specific antibodies and TGF-β3 mRNA by in situ hybridization in the nervous system of the 3- to 15-day-old chick embryo with special reference to spinal cord, hindbrain, and dorsal root ganglia (DRG). At embryonic day (E) 3, TGF-β3 mRNA as well as TGF-β2 and 3 immunoreactivities (IRs) were most prominent in the notochord, wall of the aorta, and dermomyotome. At E5 and E7, strong TGF-β2 and 3 IR were seen in or on radial glia of spinal cord and hindbrain. Radial glia in the floor plate region and ventral commissure gave the most intense signal. In the DRG, fiber strands of intense IRs representing extracellular matrix or satellite cells were seen. Neuronal perikarya did not become IR for TGF-β2 and 3 until E11, but even then the moderate signals for TGF-β3 mRNA could not be specifically localized to the neuronal cell bodies. In E11 and older embryos, spinal cord glial or glial progenitor cells, but not neuronal cell bodies were labeled for TGF-β3 mRNA. Immunocytochemistry and western blot analysis indicated that E8 DRG neurons have the TGF-β receptor type II, and treatment of these cells with NGF induces expression of TGF-β3 mRNA. The TGF-β isoforms 1, 2, and 3 did not promote survival of E8 DRG neurons in dissociated cell cultures. All three TGF-β isoforms, however, promoted neurite growth from E8 DRG explants, but were less potent than nerve growth factor. Our data suggest identical localizations of TGF-β2 and -β3 IR in the developing chick and mammalian nervous systems, underscoring the general importance of TGF-βs in fundamental events of neural development. © 1996 John Wiley & Sons, Inc.     

Expression of transforming growth factor-β isoforms in the rat male accessory sex organs and epididymis

We studied the expression and distribution of transforming growth factor-β (TGF-β) isoforms in the rat male accessory sex glands and the epididymis. Our data demonstrate the expression of both TGF-β1 and -β3 isoforms in ventral prostate (VP), seminal vesicle (SV), coagulating gland (CG), and epididymis (E) by Northern blot analysis. In addition, there was differential expression of TGF-β3 in the three regions of epididymis, the corpus region being the highest. Immunostaining data showed intense staining for latent TGF-β1 in all the male accessory glands. In contrast, no staining using antibodies specific for active TGF-β1 was observed. No expression of TGF-β2 was evident either by immunohistochemistry or Northern blot analysis. The presence of mature TGF-β3 protein was observed in the secretory epithelium of VP, CG, and corpus E. There was no detectable staining of TGF-β3 in the seminal vesicle and caput and cauda regions of epididymis. These data suggest possible differential regulation of TGF-β isoform expression in the male reproductive system and predict unique roles for individual TGF-β isoforms in sperm maturation and maintenance.     

Protein profiling of anastomosed facial nerve treated with mesenchymal stromal cells

Background aimsThe types of proteins released from mesenchymal stromal cells (MSC) are still unclear. Our aim was to compare apoptosis scores and the expression of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), neural cell adhesion molecule (NCAM)-1,matrix metalloproteinase (MMP)-1A, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-1/MMP-1A ratio, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin (NT)-3, NT-4, glial cell-derived neurotropic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-1, platelet-derived growth factor (PDGF)-α and transforming growth factor (TGF)-β1 in anastomosed facial nerves that had been treated with or without MSC.MethodsIn seven rats, the buccal branch of the right facial nerve was transected, anastomosed and treated with MSC (anastomosed + MSC group). The left buccal branch was anastomosed only (anastomosed-only group). The left mandibular branch served as an intact nerve group. On days 18–20, the distal segments of the branches were examined in terms of expression of the mentioned proteins and apoptosis scores using polymerase chain reaction (PCR) and terminal deoxynucleotidyl transferase-mediated digoxigenin-UTP nick end labeling (TUNEL) assays.ResultsMSC application significantly increased CNTF, PDGF-α, LIF, TGF-β1, BDNF and NT-3 expression (P < 0.05). MAG expression slightly decreased whereas NCAM-1, MMP-1A and FGF-2 slightly increased(P > 0.05). Changes in other proteins and apoptosis scores were not significant.ConclusionsThese results suggest that MSC increases expression of CNTF, PDGF-α, LIF,TGF-β1, BDNF and NT-3. MAG, NCAM-1, MMP-1A and FGF-2 expressions were slightly changed in this stage of nerve regeneration. The comparison of apoptotic activity was not conclusive. Overall, it appears that MSC might have differential effects on the mentioned tissue-related proteins and trophic/growth factors.     

Transforming Growth Factor Beta (TFG-β) Concentration Isoforms are Diminished in Acute Coronary Syndrome

Acute coronary syndrome (ACS) is the leading cause of death in elderly patients worldwide. Due its participation in apoptosis, fibrosis, and angiogenesis, transforming growth factor-β (TGF-β) isoforms had been categorized as risk factors for cardiovascular diseases. However, due their contradictory activities, a cardioprotective role has been suggested. The aim was to measure the plasma levels of TGF-β1, 2, and 3 proteins in patients with ACS. This was a case–control study including 225 subjects. The three activated isoforms were measured in serum using the Bio-Plex Pro TGF-β assay by means of magnetic beads; the fluorescence intensity of reporter signal was read in a Bio-Plex Magpix instrument. We observed a significant reduction of the three activated isoforms of TGF-β in patients with ACS. The three TGF-β isoforms were positively correlated with each other in moderate-to-strong manner. TGFβ-2 was inversely correlated with glucose and low-density lipoprotein (LDL)-cholesterol, whereas TGF-β3 was inversely correlated with the serum cholesterol concentration. The production of TGF-β1, TGF-β2, and TGF-β3 are decreased in the serum of patients with ACS. Further follow-up controlled studies with a larger sample size are needed, in order to test whether TGF-β isoforms could be useful as biomarkers that complement the diagnosis of ACS.     

Neuroprotective properties of ciliary neurotrophic factor for cultured adult rat dorsal root ganglion neurons

We observed that recombinant ciliary neurotrophic factor (CNTF) enhanced survival and neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. Among other neurotrophic factors (NGF and GDNF) and interleukin (IL)-6 cytokine members [IL-6, LIF, cardiotrophin-1, and oncostatin M (OSM)] at the same concentration (50 ng/ml), CNTF, as well as LIF and OSM, displayed high efficacy for the promotion of the number of viable neurons and neurite-bearing cells. CNTF enhanced the number of neurite-bearing cells in both small neurons (soma diameter <30 mum) and large neurons (soma diameter >/=30 mum), whereas NGF and GDNF promoted that in only small neurons. Western blot analysis revealed that CNTF induced phosphorylation of STAT3, Akt, and ERK1/2 in the neurons. Furthermore, the neurite outgrowth-promoting activity of CNTF was diminished by co-treatment with Janus kinase (JAK) 2 inhibitor, AG490; STAT3 inhibitor, STA-21; phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor, LY294002; and mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, in a concentration-dependent manner. Its survival-promoting activity was also affected by AG490, STA-21, and LY294002 at higher concentrations, but not by PD98059. These findings suggest the involvement of JAK2/STAT3, PI3K/Akt, and MEK/ERK signaling pathways in CNTF-induced neurite outgrowth, where the former two pathways are thought to play major roles in mediating the survival response of neurons to CNTF.     

Implication of TGF-β as a survival factor during tumour development

Transforming growth factor (TGF)-β is a pleiotropic secretory protein which inhibits and potentiates tumour progression during early and late stage of tumourigenicity, respectively. However, it still remains veiled how TGF-β signalling reveals its two faces. Hoshino et al. (Autocrine TGF-β protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim, J. Biochem. 2011;149:55-65) demonstrated a new aspect of TGF-β as a survival factor in highly metastatic breast cancer cells from which TGF-β1 and TGF-β3 are abundantly expressed. They found that TGF-β suppressed the expression of BH3-only protein Bim which promotes programmed death signalling via release of cytochrome c from mitochondria. Further interestingly, forkhead box C1 (Foxc1) whose expression is suppressed upon TGF-β stimulation is involved in the expression of Bim. Based on their results, autocrine TGF-β signalling in certain breast cancers promotes cell survival via inhibition of apoptotic signalling. Thus, the inhibitors for activin receptor-like kinase (ALK)5 kinase might exert a curative influence on certain types of metastatic breast cancers.     

Transforming Growth Factor-β Isoform Expression in Mature Human Healthy and Carious Molar Teeth

Transforming growth factor (TGF)-β isoforms have been implicated in cellular signalling during tooth development and repair, but little is known of their cellular localisation or distribution within the dental tissues in the mature tooth. This study investigated the presence of TGF-β1, β2 and β3 isoforms in tissues of sound and carious human molar teeth, to understand better the expression of TGF-βs during health and disease. In healthy tissues, odontoblasts, cells of the cell rich layer, pulpal fibroblasts and endothelial cells were stained to varying degrees for all isoforms, with TGF-β3 showing the greatest intensity and TGF-β1 the weakest intensity. Similar patterns of staining were observed in carious teeth; however, TGF-β1 showed significantly increased staining intensity within odontoblasts and pulpal cells of carious teeth (p<0.001). Biochemical analysis showed greater amounts of TGF-β1 in tertiary dentine than in primary dentine samples. The expression of TGF-βs in odontoblasts and the increased presence of TGF-β1 in tertiary dentine suggest that these isoforms may be important in odontoblast behaviour and the modulation of the tissue response to injury.     

Prevention of apoptosis in CNTF-dependent neurons by a mutant ICE and by viral protein CrmA but not by proto-oncogene product Bcl-2

The interleukin-1beta converting enzyme (ICE) gene family, (homologues of C. elegans cell death gene product Ced-3) plays an important role in controlling programmed cell death. Nerve growth factor (NGF) promotes survival of cultured embryonic chicken dorsal root ganglion neurons. Ciliary ganglion neurons depend exclusively on ciliary neurotrophic factor (CNTF) for survival. Complete depletion of NGF or CNTF from culture medium induces apoptosis in both types of neurons. We can prevent apoptosis, due either to NGF or CNTF withdrawal and in either type of neuron, by overexpression of a mutant inactive ICE and an ICE inhibitor, the product of cowpox virus gene crmA. Bcl-2 does not prevent apoptosis in CNTF-dependent ciliary neurons or DRG neurons as it does in NGF-dependent neurons. These results suggest that neuronal cell death is mediated through a common effector mechanism involving the Ice family of genes, whereas different suppression mechanisms are engaged depending upon the specific neurotrophic factors present.     

Neuronal survival in intact ciliary ganglia in vivo and in vitro: Ciliary neuronotrophic factor as a target surrogate

Ciliary neuronotrophic factor (CNTF) requirements for neuronal survival in the intact ciliary ganglion (CG) have been investigated in organ culture. Exogenous CNTF was not essential for neuronal survival until embryonic Day 8. Three-day cultures from 5-day ganglia were similar with or without CNTF, showing numerous neurons and extensive neuritic development. In 3-day cultures from 8-day-old ganglia, however, no neurons survived without CNTF, and the ganglia contained only nonneuronal cells and cell debris. Similar ganglia cultured with CNTF contained many neurons, surrounded by nonneuronal cells, and abundant neuritic processes. Morphologic maturation of the neurons was less advanced in CNTF-supported ganglia than in their in vivo counterparts.     

Expression and histochemical localization of ciliary neurotrophic factor in cultured adult rat dorsal root ganglion neurons

Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not in neurons. After peripheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival and axonal regeneration. In the present study, we examined the expression and histochemical localization of CNTF in adult rat DRG in vivo and in vitro. In contrast to the restricted expression in Schwann cells in vivo, we observed abundant CNTF mRNA and protein expression in DRG neurons after 3 h, 2, 7, and 15 days in dissociated cell culture. At later stages (7 and 15 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and regenerating neurites. These results suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF immunoreactivity in DRG neurons in explant culture, disruption of cell–cell interactions, rather than the culture itself, may be an inducible factor for localization of CNTF in the neurons.     

TGF-β isoforms inhibit hepatitis C virus propagation in transforming growth factor beta/SMAD protein signalling pathway dependent and independent manners

Transforming growth factor beta (TGF-β) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-β isoforms, including TGF-β1, TGF-β2 and TGF-β3, remain unclear. Here, we demonstrated that all of the three TGF-β isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-β isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-β isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-β/SMAD signalling pathway–dependent and TGF-β/SMAD signalling pathway–independent manners. TGF-β isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-β1 and TGF-β2, not TGF-β3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-β isoforms in the HCV-related liver disease progression.     

Sympathetic neuronal survival induced by retinal trophic factors

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti‐NGF (1 μg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1–50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin‐receptors TrkA, TrkB, or TrkC had no effect on RCM‐induced sympathetic neuron survival. The survival effects were not blocked by anti‐GDNF, anti‐TGFβ, and anti‐CNTF and were not mimicked by FGFb (0.1–10 nM). LY294002 at 50 μM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high‐molecular weight retinal secreted factor that supports sympathetic neurons in culture. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 13–23, 2002     

Membrane-associated neurotransmitter stimulating factor is very similar to ciliary neurotrophic factor.

Membrane-associated neurotransmitter stimulating factor (MANS) can modulate sympathetic neurotransmitter expression and promote ciliary neuron survival in cell culture. Previous studies have shown that its biological effects and biochemical properties are similar to those of ciliary neurotrophic factor (CNTF). In addition, CNTF is present in spinal cord, the source of MANS. These observations raised the possibility that MANS preparations contain CNTF. We find that partially purified MANS fractions contain a 24-kD protein that is recognized in Western blots by an antiserum generated against recombinant rat CNTF (rCNTF). This antiserum immunoprecipitates virtually all the cholinergic-inducing and the ciliary neurotrophic activities present in MANS preparations. When iodinated rCNTF is incubated with spinal cord membranes, a significant proportion of the labeled CNTF segregates with the membrane pellet. The membrane-associated exogenous CNTF can be eluted from the membrane fraction by treatment with high-salt solutions, similar to that used to solubilize MANS from spinal cord membranes. Our data suggest that a substantial portion of the cholinergic differentiation and ciliary neurotrophic activities present in MANS preparations can be attributed to CNTF or a CNTF-like molecule.     

Promotion of survival and neurite outgrowth of cultured peripheral neurons by exogenous lipids and detergents

Gangliosides, in particular the monosialoglycosphingolipids Gtet 1 (GM1), have previously been implicated in the mediation of neuronal rescue and restitutional axonal growth, both in vitro and subsequent to brain and peripheral nerve lesions. In the present study it is shown that the bis-sialosyl gangliosides Gtet2b and Gtet3b, but not the gangliosides Gtet2a and Gtet1, promote the survival of dissociated dorsal root ganglion (DRG) neurons cultured from Embryonic Day (E) 8 chicks (DRG8) almost to the same extent as nerve growth factor (NGF). Ciliary ganglion (CG) neurons from E8 chicks (CG8) and DRG10 neurons were virtually not supported suggesting considerable specificity in terms of neuronal targets and developmental stages being addressed. Moreover, a variety of other lipids including cerebroside (Cb), dipalmitoylphosphatidylcholine (DPPC) and -serine (DPPS), sulfatide (Sf), and sphingomyelin (Sm) were tested for putative survival promoting activity toward chick CG, DRG, and lumbar sympathetic ganglion (SG11) neurons. At the highest concentration employed (2.5 x 10(-5) M), Sm, DPPC, and DPPS maintained between 45 and 65% of the plateau survival with CG8 (maximally supported by ciliary neuronotrophic factor (CNTF], DRG8, and DRG10 neurons, and 30 to 40% with SG11 neurons. Cb supported CG8 neurons at about 55% of the plateau value achieved with CNTF, but had hardly any effect on the other neuron populations tested. Control experiments using highly enriched neurons and serum-free conditions assured that the effects were unlikely to be mediated by serum components or nonneuronal cells. A variety of detergents, in particular Triton X-100, also promoted the survival of CG8 and DRG10 neurons. Ganglioside Gtet1, Sm, and Triton X-100 shifted the NGF titration curve for DRG10 neurons between 6- and 15-fold in a dose-dependent manner suggesting synergisms between NGF and lipids for neuronal maintenance. These results document the neuronotrophic potency of certain gangliosides, a heterogeneous group of structurally unrelated lipids, and detergents. The mechanisms by which these agents modulate neuronal survival still await clarification.     

The FGFR1 inhibitor PD 173074 selectively and potently antagonizes FGF-2 neurotrophic and neurotropic effects

Basic fibroblast growth factor (FGF-2) promotes survival and/or neurite outgrowth from a variety of neurons in cell culture and regenerative processes in vivo. FGFs exert their effects by activating cell surface receptor tyrosine kinases. FGF receptor (FGFR) inhibitors have not been characterized on neuronal cell behaviors to date. In the present study, we show that the FGFR1 inhibitor PD 173074 potently and selectively antagonized the neurotrophic and neurotropic actions of FGF-2. Nanomolar concentrations of PD 173074 prevented FGF-2, but not insulin-like growth factor-1, support of cerebellar granule neuron survival under conditions of serum/K(+) deprivation; another FGF-2 inhibitor, SU 5402, was effective only at a 1,000-fold greater concentration. Neither PD 173074 nor SU 5402, at 100 times their IC(50) values, interfered with the survival of dorsal root ganglion neurons promoted by nerve growth factor, ciliary neurotrophic factor, or glial cell line-derived neurotrophic factor. PD 173074 and SU 5402 displayed 1,000-fold differential IC(50) values for inhibition of FGF-2-stimulated neurite outgrowth in PC12 cells and in granule neurons, and FGF-2-induced mitogen-activated protein kinase (p44/42) phosphorylation. The two inhibitors failed to disturb downstream signalling stimuli of FGF-2. PD 173074 represents a valuable tool for dissecting the role of FGF-2 in normal and pathological nervous system function without compromising the actions of other neurotrophic factors.     

CNTF对烧伤大鼠血清引起大鼠海马神经元细胞毒性的影响

应用整体和离体神经元培养,观察CNTF对烧伤大鼠海马神经元及烧伤血清引起海马神经元损伤的影响,结果表明,大鼠烧伤后海马组织神经元数目减少,NO含量升高;烧伤大鼠血清可引起培养的海马神经元细胞存活率下降,培养液中NO含量升高;CNTF能降低烧伤大鼠海马组织中NO的含量,保护海马神经元,并能提高培养的海马神经元的存活率,减少培养液中NO含量,其作用呈剂量依赖性;CNTF对神经元存活率的影响与NO含量呈显著负相关,提示CNTF对烧伤大鼠血清引起的海马神经元损伤有保护作用,其作用机制可能是通过抑制NO的神经毒性。