Effects Of Ciliary Neurotrophic Factor Research Articles

CNTF Attenuates Vasoproliferative Changes Through Upregulation of SOCS3 in a Mouse-Model of Oxygen-Induced Retinopathy.

PurposeRetinal vascular disease represents a major cause for vision loss in the Western world. Recent research has shown that neuronal and vascular damage are closely related in retinal disease. Ciliary neurotrophic factor (CNTF) is a well-studied neurotrophic factor that is currently being tested in clinical trials for the treatment of retinal degenerative diseases and macular telangiectasia. However, little is known about its effect on retinal vasculature. In this study, we investigate the effects of CNTF in retinal neovascular disease using the mouse model of oxygen-induced retinopathy (OIR).MethodsNewborn pups were exposed to 75% oxygen from postnatal day (P)7 to P12 and subsequently returned to room air. Ciliary neurotrophic factor was injected intravitreally at OIR P12 and the vaso-obliterated and neovascular areas were quantified at OIR P17. Immunohistochemistry, RNA, and protein analysis were used to identify CNTF-responsive cells. In vitro experiments were performed to analyze the effect of CNTF on endothelial and astroglial cells.ResultsIn the OIR model, CNTF facilitated capillary regrowth and attenuated preretinal neovascularization in a dose-dependent manner. The protective effect of CNTF was mediated via activation of the JAK/STAT3/SOCS3 signaling pathway. Immunohistochemical studies identified endothelial cells among others as CNTF-responsive cells in the retina. In vitro studies confirmed the anti-angiogenic effect of CNTF on endothelial cell sprouting.ConclusionsThis study provides evidence for a therapeutic potential of CNTF beyond degenerative retinal disease. Vasoproliferative retinopathies may benefit from a CNTF-dependent and SOCS3-mediated angiomodulatory effect.

Ciliary Neurotrophic Factor Promotes the Migration of Corneal Epithelial Stem/progenitor Cells by Up-regulation of MMPs through the Phosphorylation of Akt

The migration of limbal epithelial stem cells is important for the homeostasis and regeneration of corneal epithelium. Ciliary neurotrophic factor (CNTF) has been found to promote corneal epithelial wound healing by activating corneal epithelial stem/progenitor cells. However, the possible effect of CNTF on the migration of corneal epithelial stem/progenitor cells is not clear. This study found the expression of CNTF in mouse corneal epithelial stem/progenitor cells (TKE2) to be up-regulated after injury, on both gene and protein level. CNTF promoted migration of TKE2 in a dose-dependent manner and the peak was seen at 10 ng/ml. The phosphorylation level of Akt (p-Akt), and the expression of MMP3 and MMP14, were up-regulated after CNTF treatment both in vitro and in vivo. Akt and MMP3 inhibitor treatment delayed the migration effect by CNTF. Finally, a decreased expression of MMP3 and MMP14 was observed when Akt inhibitor was applied both in vitro and in vivo. This study provides new insights into the role of CNTF on the migration of corneal epithelial stem/progenitor cells and its inherent mechanism of Up-regulation of matrix metalloproteinases through the Akt signalling pathway.

Ciliary neurotrophic factor upregulates follistatin and Pak1, causes overexpression of muscle differentiation related genes and downregulation of established atrophy mediators in skeletal muscle

IntroductionThe Ciliary Neurotrophic Factor (CNTF) is a pluripotent cytokine with anorexigenic actions in the hypothalamus that improves insulin sensitivity, increases energy expenditure and induces weight loss. Since CNTF also has an established myotrophic role, we sought to examine whether skeletal muscle contributes to the CNTF-induced metabolic improvement and identify the molecular mechanisms mediating these effects. MethodsWe used a mouse model of diet-induced obesity, to which high or low CNTF doses were administered for 7days. Whole transcriptome expression levels were analyzed in dissected soleus muscles using microarrays and data were then confirmed using qRT-PCR. ResultsWe demonstrate that CNTF administration significantly downregulates leptin, while it upregulates follistatin and Pak1; a molecule associated with insulin sensitization in skeletal muscle. A significant overexpression of muscle differentiation related genes and downregulation of established atrophy mediators was observed. ConclusionsThe overall gene expression changes suggest an indirect, beneficial effect of CNTF on metabolism, energy expenditure and insulin sensitivity, exerted by the pronounced stimulation of muscle growth, with similarities to the described effect of follistatin and the activation of the Akt pathway in skeletal muscle.

Synergetic effects of ciliary neurotrophic factor and olfactory ensheathing cells on optic nerve reparation (complete translation).

At present, there is no effective treatment for the repair of the optic nerve after injury, or improvement of its microenvironment for regeneration. Intravitreally injected ciliary neurotrophic factor (CNTF) and olfactory ensheathing cells (OECs) promote the long-distance regrowth of severed optic nerve fibers after intracranial injury. Here, we examined the efficacy of these techniques alone and in combination, in a rat model of optic nerve injury. We injected condensed OEC suspension at the site of injury, or CNTF into the vitreous body, or both simultaneously. Retrograde tracing techniques showed that 4 weeks postoperatively, the number of surviving retinal ganglion cells and their axonal density in the optic nerve were greater in rats subjected to OEC injection only than in those receiving CNTF injection only. Furthermore, combined OEC + CNTF injection achieved better results than either monotherapy. These findings confirm that OECs are better than CNTF at protecting injured neurons in the eye, but that combined OEC and CNTF therapy is notably more effective than either treatment alone.

Intravitreal Ciliary Neurotrophic Factor Transiently Improves Cone-Mediated Function in a CNGB3-/- Mouse Model of Achromatopsia.

Ciliary neurotrophic factor (CNTF) was recently shown to augment cone function in CNGB3 mutant achromat dogs. However, testing CNTF-releasing implant in human CNGB3 achromats failed to show benefit. We evaluated the effects of CNTF protein on the retinal function in an additional achromatopsia model, the CNGB3-/- mouse. Fifty-nine CNGB3-/- mice (postnatal day [PD] ± SD = 30 ± 7) received a unilateral intravitreal injection of 1 or 2 μg CNTF protein, and 15 wild-type (WT) mice (PD = 34 ± 3) received 1 μg CNTF. Retinal function was evaluated by flash ERG and photopic flicker ERG (fERG) at 7 and 14 days after treatment. Seven days post CNTF, the photopic b-wave Vmax was significantly increased in CNGB3-/- mice (P < 0.01), whereas it was reduced in WT mice (P < 0.05). Ciliary neurotrophic factor significantly increased the amplitude of photopic fERG and the photopic oscillatory potentials (OPs) in CNGB3-/- mice. Ciliary neurotrophic factor did not alter the scotopic a-wave in either CNGB3-/- or WT mice, but it increased the scotopic b-wave k (P < 0.01) in CNGB3-/- mice, indicating diminished scotopic sensitivity, and reduced the scotopic b-wave Vmax in WT mice (P < 0.05). No difference was found in ERG parameters between 1 or 2 μg CNTF. Fourteen days after CNTF injection the ERG changes in CNGB3-/- mice were lost. Intravitreal bolus CNTF protein caused a small and transient improvement of cone-mediated function in CNGB3-/- mice, whereas it reduced rod-mediated function. The increase in photopic OPs and the lack of changes in scotopic a-wave suggest a CNTF effect on the inner retina.

The trophic effect of ciliary neurotrophic factor on injured masseter muscle in rat.

Occlusal trauma is one of the most common forms of oral biting dysfunction. Long-term occlusal trauma could weaken the stomatognathic system; especially damage one's masticatory muscle. Through using the rat model, this study investigated the trophic effect of ciliary neurotrophic factor (CNTF) on injured masseter muscle. Male Wistar rats (n=36) were randomly divided into five experimental groups and one control group (6 rats per group). Animals in the experimental group were cemented modified crowns on their mandibular first molars to artificially induce occlusal trauma in 1, 3, 7, 14, and 28 days. Control group was sham-treated with forced mouth-opening for about 5 min, while no crowns were placed. After 28 days of treatment, all rats were euthanized and their masseter muscle was collected. Through immunofluorescence and real-time quantitative PCR, the expression of desmin, CNTF, and CNTFRα was investigated in rat masseter muscle. The microstructure of masseter muscle was observed by transmission electron microscope. The expression of desmin showed a time-dependent decrease on traumatic and non-traumatic sides masseter, until reached the nadir at the 14(th) day, then restored to its normal level at the 28(th) day; however, the expression of CNTF and CNTFRα on the traumatic and non-traumatic sides increased from day 7, reached the peak at the 14(th) day, and returned to normal level on the 28(th) day. CNTF, as an important neurotrophic factor, was tightly associated to the restoring of rat injured masseter muscle, which provides new target and treatment method for clinical application.

The neuroprotective agent CNTF decreases neuronal metabolites in the rat striatum: an in vivo multimodal magnetic resonance imaging study.

Ciliary neurotrophic factor (CNTF) is neuroprotective against multiple pathologic conditions including metabolic impairment, but the mechanisms are still unclear. To delineate CNTF effects on brain energy homeostasis, we performed a multimodal imaging study, combining in vivo proton magnetic resonance spectroscopy, high-performance liquid chromatography analysis, and in situ glutamate imaging by chemical exchange saturation transfer. Unexpectedly, we found that CNTF expression through lentiviral gene transfer in the rat striatum significantly decreased the levels of neuronal metabolites (N-acetyl-aspartate, N-acetyl-aspartyl-glutamate, and glutamate). This preclinical study shows that CNTF remodels brain metabolism, and suggests that decreased levels of neuronal metabolites may occur in the absence of neuronal dysfunction.

Action of the ciliary neurotrophic factor in mouse brainstem

Systemic administration of ciliary neurotrophic factor (CNTF) determines sustained weight loss in rodents and humans. The anorectic effect of CNTF is due, at least in part, to activation of the signal transducer and activator of transcription (STAT) 3, STAT1 and STAT5 in neuronal and glial cells of the tuberal hypothalamus (Severi et al., 2012, 2013 and unpublished data). To assess whether CNTF also exerts an action on the brainstem feeding centres, male Swiss CD-1 mice were intraperitoneally treated with rat recombinant CNTF (0.3 mg/kg of body weight; CNTF-treated mice) or with saline (control mice) for 45 minutes (min) to perform P-STAT3, P-STAT1 and P-STAT5 immunohistochemistry, and for 120 min to detect c-Fos expression. Double staining experiments with neuronal and glial markers and confocal microscopy analyses were performed to assess the phenotype of the CNTF-responsive cells. P-STAT3-, P-STAT1- or P-STAT5-positive cells were not detected in brainstem coronal sections from control mice. In contrast, CNTF administration led to a strong activation of the transcription factors STAT3, STAT1 and STAT5 in CNTF receptor α-bearing glial cells of the area postrema, a circumventricular organ devoid of blood-brain barrier, that allows circulating molecules to gain access to the adjacent brain parenchyma. In 120 min CNTF-treated mice specific nuclear staining for c-Fos was detected not only in cells of the area postrema but also in numerous neurons widely distributed in the nucleus of the solitary tract (Sol), the dorsal motor nucleus of vagus (DMV) and parabrachial nuclei. Ongoing experiments on capsaicin-treated (deafferented) mice will disclose a possible vagal contribution to the Sol and DMV neuronal activation due to a peripheral action of CNTF in the gastro-intestinal tract. Collectively, these data show for the first time that CNTF similarly to other satiety factor, like leptin, has redundant actions on both hypothalamic and brainstem energy balance neuronal networks. Unravelling the phenotype of brainstem neurons responsive to CNTF will clarify its role in distinctive aspects of feeding behaviour.

CNTF regulates neurite outgrowth and neuronal migration through JAK2/STAT3 and PI3K/Akt signaling pathways of DRG explants with gp120-induced neurotoxicity in vitro

HIV envelope glycoprotein gp120 is highly involved in HIV infection-related peripheral neuropathy, but its mechanism remains incompletely understood. The therapy of this neuropathy is still a big clinical challenge for neurologists. The organotypically cultured dorsal root ganglion (DRG) explants were used to test the neurotoxic actions of gp120 and the therapeutic effects of ciliary neurotrophic factor (CNTF) on gp120-induced neurotoxicity. The results showed that gp120 inhibited neurite outgrowth and neuronal migration from the DRG explants in a dose-dependent manner. HIV-gp120 also inhibited growth-associated protein 43 (GAP-43) expression and induced apoptosis of the migrating neurons. CNTF improved neurite outgrowth, neuronal migration, and GAP-43 expression inhibited by gp120. CNTF also rescued neuronal apoptosis induced by gp120. Either Janus kinase 2 (JAK2) inhibitor AG490 or phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002 blocked the effects of CNTF. These data imply that CNTF improved neuronal status by promoting GAP-43 expression and inhibiting apoptosis through JAK2/signal transducer and activator of transcription 3 (STAT3) and PI3K/Akt signaling pathways of DRG neurons with gp120-induced neurotoxicity. These data offered a new clue for elucidating the mechanisms of HIV infection-related peripheral neuropathy and facilitating the development of novel therapy.

Ciliary neurotrophic factor reverses aberrant mitochondrial bioenergetics through the JAK/STAT pathway in cultured sensory neurons derived from streptozotocin-induced diabetic rodents.

Mitochondrial dysfunction occurs in sensory neurons and contributes to diabetic neuropathy. Ciliary neurotrophic factor (CNTF) stimulates axon regeneration in type 1 diabetic rodents and prevents deficits in axonal caliber, nerve conduction, and thermal sensation. We tested the hypothesis that CNTF enhances sensory neuron function in diabetes through JAK/STAT (Janus kinase/signal transducers and activators of transcription) signaling to normalize impaired mitochondrial bioenergetics. The effect of CNTF on gene expression and neurite outgrowth of cultured adult dorsal root ganglia (DRG) sensory neurons derived from control and streptozotocin (STZ)-induced diabetic rodents was quantified. Polarization status and bioenergetics profile of mitochondria from cultured sensory neurons were determined. CNTF treatment prevented reduced STAT3 phosphorylation (Tyr 705) in DRG of STZ-diabetic mice and also enhanced STAT3 phosphorylation in rat DRG cultures. CNTF normalized polarization status of the mitochondrial inner membrane and corrected the aberrant oligomycin-induced mitochondrial hyperpolarization in axons of diabetic neurons. The mitochondrial bioenergetics profile demonstrated that spare respiratory capacity and respiratory control ratio were significantly depressed in sensory neurons cultured from STZ-diabetic rats and were corrected by acute CNTF treatment. The positive effects of CNTF on neuronal mitochondrial function were significantly inhibited by the specific JAK inhibitor, AG490. Neurite outgrowth of sensory neurons from age-matched control and STZ-induced diabetic rats was elevated by CNTF and blocked by AG490. We propose that CNTF's ability to enhance axon regeneration and protect from fiber degeneration in diabetes is associated with its targeting of mitochondrial function and improvement of cellular bioenergetics, in part, through JAK/STAT signaling.

Neuroprotective properties of ciliary neurotrophic factor on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells.

Ciliary neurotrophic factor (CNTF) is a neurocytokine, which could promote survival and/or differentiation in many cell types. In this study, the biological effects of CNTF on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells and the underlying molecular mechanism of this effect were investigated for the first time. The results showed that RA was able to increase cells susceptibility to CNTF via regulating the expression levels of CNTF receptors. A further study revealed that CNTF could induce phosphorylation of STAT3, Akt and ERK1/2 in RA-predifferentiated SH-SY5Y neuroblastoma cells, while the promoting activity of CNTF on survival and neurite growth of cells was attenuated by co-treatment with JAK2 inhibitor AG490 (25 μM), STAT3 inhibitor Curcumin (50 μM), PI3K inhibitor LY-294002 (50 µM), but not by co-treatment with MEK inhibitor PD98059 (50 μM). These findings suggested that JAK2/STAT3, as well as PI3K/Akt, play important roles in mediating the survival and neurite growth response of RA-predifferentiated cells to CNTF. Our study may be useful to further understand the functional role of CNTF and offer a convenient model to explore the therapeutic potential of CNTF in neurodegenerative diseases.

The Cytokine Ciliary Neurotrophic Factor (CNTF) Activates Hypothalamic Urocortin-Expressing Neurons Both In Vitro and In Vivo

Ciliary neurotrophic factor (CNTF) induces neurogenesis, reduces feeding, and induces weight loss. However, the central mechanisms by which CNTF acts are vague. We employed the mHypoE-20/2 line that endogenously expresses the CNTF receptor to examine the direct effects of CNTF on mRNA levels of urocortin-1, urocortin-2, agouti-related peptide, brain-derived neurotrophic factor, and neurotensin. We found that treatment of 10 ng/ml CNTF significantly increased only urocortin-1 mRNA by 1.84-fold at 48 h. We then performed intracerebroventricular injections of 0.5 mg/mL CNTF into mice, and examined its effects on urocortin-1 neurons post-exposure. Through double-label immunohistochemistry using specific antibodies against c-Fos and urocortin-1, we showed that central CNTF administration significantly activated urocortin-1 neurons in specific areas of the hypothalamus. Taken together, our studies point to a potential role for CNTF in regulating hypothalamic urocortin-1-expressing neurons to mediate its recognized effects on energy homeostasis, neuronal proliferaton/survival, and/or neurogenesis.

Antineuroinflammatory and neurotrophic effects of CNTF and C16 peptide in an acute experimental autoimmune encephalomyelitis rat model

Experimentalallergic encephalomyelitis (EAE) is an animal model for inflammatory demyelinating autoimmune disease, i.e., multiple sclerosis (MS). In the present study, we investigated the antineuroinflammatory/neuroprotective effects of C16, an ανβ3 integrin-binding peptide, and recombinant rat ciliary neurotrophic factor (CNTF), a cytokine that was originally identified as a survival factor for neurons, in an acute rodent EAE model. In this model, C16 peptide was injected intravenously every day for 2 weeks, and CNTF was delivered into the cerebral ventricles with Alzet miniosmotic pumps. Disease severity was assessed weekly using a scale ranging from 0 to 5. Multiple histological and molecular biological assays were employed to assess inflammation, axonal loss, neuronal apoptosis, white matter demyelination, and gliosis in the brain and spinal cord of different groups. Our results showed that the EAE induced rats revealed a significant increase in inflammatory cells infiltration, while C16 treatment could inhibit the infiltration of leukocytes and macrophages down to 2/3–1/3 of vehicle treated EAE control (P < 0.05). The delayed onset of disease, reduced clinical score (P < 0.01) in peak stage and more rapid recovery also were achieved in C16 treated group. Besides impairing inflammation, CNTF treatment also exerted direct neuroprotective effects, decreasing demyelination and axon loss score (P < 0.05 versus vehicle treated EAE control), and reducing the neuronal death from 40 to 50% to 10 to 20% (P < 0.05). Both treatments suppressed the expression of cytokine tumor necrosis factor-α and interferon-γ when compared with the vehicle control (P < 0.05). Combined treatment with C16 and CNTF produced more obvious functional recovery and neuroprotective effects than individually treatment (P < 0.05). These results suggested that combination treatment with C16 and CNTF, which target different neuroprotection pathways, may be an effective therapeutic alternative to traditional therapy.

Fibroblast growth factor-2 counteracts the effect of ciliary neurotrophic factor on spontaneous differentiation in adult hippocampal progenitor cells

Neural stem/progenitor cells (NSCs) can spontaneously differentiate into neurons and glial cells in the absence of mitogen fibroblast growth factor-2 (FGF-2) or epidermal growth factor (EGF) in medium and the spontaneous differentiation of NSCs is mediated partially by endogenous ciliary neurotrophic factor (CNTF). This study examined the relationship of FGF-2 and CNTF in the spontaneous differentiation of adult hippocampal progenitor cells (AHPs). AHPs were cultured in the medium containing different concentration of FGF-2 (1-100 ng/mL). Western blotting and immunofluorescence staining were applied to detect the expression of the astrocytic marker GFAP, the neuronal marker Tuj1, the oligodendrocytic marker CNPase and, Nestin, the marker of AHPs. The expression of endogenous CNTF in AHPs at early (passage 4) and late stage (passage 22) was also measured by Western blotting. The results showed that FGF-2 increased the expression of Nestin, dramatically inhibited the expression of GFAP and Tuj1 and slightly suppressed the expression of CNPase. FGF-2 down-regulated the expression of endogenous CNTF in AHPs at both early (passage 4) and late stage (passage 22). These results suggested that FGF-2 could inhibit the spontaneous differentiation of cultured AHPs by negatively regulating the expression of endogenous CNTF in AHPs.

Abstract 18960: Ciliary Neurotrophic Factor Increases the Angiogenic Properties of Human Pluripotent Stem Cell Derived Neuropilin-1+ CD34+ Nascent Endothelial Cells Via a JAK2 Dependent Pathway

Pluripotent stem cells are a model to identify novel regulators of angiogenesis. Previously, we showed that Neuropilin-1 (NRP1) CD34- cells were vascular precursors. Subsequently, genomic analysis revealed that NRP1+ CD34+ cells had transcript content consistent with nascent endothelium, and unique growth factor receptors that may be exploited to modulate their angiogenic properties. The objectives of our study was to verify that NRP1+ CD34+ cells form endothelium, and determine the function of the Ciliary Neurotrophic Factor (CNTF) Receptor and its downstream kinase Jak2 in angiogenesis. Methods and Results: Human induced pluripotent stem cells (hiPSCs) were differentiated as embyroid bodies and NRP1+ CD34+ nascent endothelial cells were sorted. NRP1+ CD34+ cells readily formed sheets of endothelium expressing VE-Cadherin, CD146, PECAM, and eNOS, absorbed acetylated LDL, and formed tubule networks in Matrigel. These features confirmed their endothelial properties. Here we show that CNTF promotes angiogenic sprouting of NRP1+ CD34+ cells via a Jak2 dependent mechanism. RT-PCR analysis of NRP1+ CD34+ transcripts showed a significant (&gt; 5 fold) increase in the growth factor receptor CNTFR, and the downstream signaling partners Jak2, Stat5A and Stat6 versus undifferentiated hiPSCs. CNTF increased the length of endothelial tubules in Matrigel 44±7.3% vs. no CNTF (p≤0.05), while inhibition of JAK2 in the presence of CNTF blocked the effect of CNTF. Conclusions: NRP1+ CD34+ cells derived from human pluripotent stem cells readily form endothelium, and their angiogenic properties can be augmented by activation of a novel CNTFR-Jak2 mediated pathway. Collectively, the findings confirm that NRP1+ CD34+ cells are a nascent endothelial cell, and identify a novel growth factor signaling pathway involved in angiogenesis, suggesting a role for CNTF in promotion of angiogenesis in therapy for vascular diseases.

Local axonal function of STAT3 rescues axon degeneration in the pmn model of motoneuron disease

Axonal maintenance, plasticity, and regeneration are influenced by signals from neighboring cells, in particular Schwann cells of the peripheral nervous system. Schwann cells produce neurotrophic factors, but the mechanisms by which ciliary neurotrophic factor (CNTF) and other neurotrophic molecules modify the axonal cytoskeleton are not well understood. In this paper, we show that activated signal transducer and activator of transcription-3 (STAT3), an intracellular mediator of the effects of CNTF and other neurotrophic cytokines, acts locally in axons of motoneurons to modify the tubulin cytoskeleton. Specifically, we show that activated STAT3 interacted with stathmin and inhibited its microtubule-destabilizing activity. Thus, ectopic CNTF-mediated activation of STAT3 restored axon elongation and maintenance in motoneurons from progressive motor neuronopathy mutant mice, a mouse model of motoneuron disease. This mechanism could also be relevant for other neurodegenerative diseases and provide a target for new therapies for axonal degeneration.

The cytokines cardiotrophin-like cytokine/cytokine-like factor-1 (CLC/CLF) and ciliary neurotrophic factor (CNTF) differ in their receptor specificities

Ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC) are two cytokines with neurotrophic and immunomodulatory activities. CNTF is a cytoplasmic factor believed to be released upon cellular damage, while CLC requires interaction with a soluble cytokine receptor, cytokine-like factor 1 (CLF), to be efficiently secreted. Both cytokines activate a receptor complex comprising the cytokine binding CNTF receptor α (CNTFRα) and two signaling chains namely, leukemia inhibitory factor receptor β (LIFRβ) and gp130. Human CNTF can recruit and activate an alternative receptor in which CNTFRα is substituted by IL-6Rα. As both CNTF and CLC have immune-regulatory activities in mice, we compared their ability to recruit mouse receptors comprising both gp130 and LIFRβ signaling chains and either IL-6Rα or IL-11Rα which, unlike CNTFRα, are expressed by immune cells. Our results indicate that 1) mouse CNTF, like its human homologue, can activate cells expressing gp130/LIFRβ with either CNTFRα or IL-6Rα and, 2) CLC/CLF is more restricted in its specificity in that it activates only the tripartite CNTFR. Several gp130 signaling cytokines influence T helper cell differentiation. We therefore investigated the effect of CNTF on CD4 T cell cytokine production. We observed that CNTF increased the number of IFN-γ producing CD4 T cells. As IFN-γ is considered a mediator of the therapeutic effect of IFN-β in multiple sclerosis, induction of IFN-γ by CNTF may contribute to the beneficial immunomodulatory effect of CNTF in mouse multiple sclerosis models. Together, our results indicate that CNTF activates the same tripartite receptors in mouse and human cells and further validate rodent models for pre-clinical investigation of CNTF and CNTF derivatives. Furthermore, CNTF and CLC/CLF differ in their receptor specificities. The receptor α chain involved in the immunomodulatory effects of CLC/CLF remains to be identified.

P162 Ciliary neurotrophic factor protects mice against type 1 diabetes and interleukin-1B-induced beta-cell apoptosis through SOCS3 expression: Role of stat1/stat3 balance in cytokine- induced iNOS-dependent beta-cell death

Introduction Type 1 Diabetes (DM1) is characterized by a cytokine-dependent loss of pancreatic islet beta-cell mass with subsequent hyperglycaemia. Ciliary Neurotrophic Factor (CNTF) protects pancreatic islets against cytokine-induced apoptosis and increases SOCS3 expression. In addition, beta-cell apoptosis seems to be induced by STAT1 and inhibited by STAT3 activation. We evaluated if CNTF protects mice against Streptozotocin-induced Type 1 Diabetes, if this protection is dependent of SOCS3 and/or iNOS expression, and what the role of STAT1/STAT3 activation in CNTF effects on cytokine-induced iNOS-dependent beta-cell apoptosis. Methods We developed SOCS3 Knockdown mice (SOCS3Kd) by Antisense technique from a C57BL6 Wild-Type background. To evaluate the role of iNOS on the CNTF protective effects in vivo we used iNOS-knockout mice. For Streptozotocin-induced Type 1 Diabetes, 4–6 weeks-old mice were treated or not with 0.1 mg/kg of CNTF for 5 days, receiving, intraperitoneally, 80 mg/kg of Streptozotocin (STZ) on the 5th day. Glycaemia from non-fasted mice was measured at days 0, 1, 7, 14, 21, and 28 after STZ. DM1 was considered as blood glycaemia >11.2 mmol/l for 2 days. We evaluated mice glycaemia, DM1 incidence, time to DM1 onset and DM1 severity. Pancreatic islets were isolated by collagenase method for mRNA and protein extraction. SOCS3 was inhibited in MIN6 cells using an SOCS3 specific siRNA (SOCS3kd cells), and iNOS inhibition was obtained by treating MIN6 cells with 1 mmol/l of the iNOS inhibitor LMEM (iNOSkd cells). Wild-type (WT), SOCS3kd, and iNOSkd cells were cultured with 10 ug/ml of IL1beta, 1 nmol/l CNTF or both. mRNA levels were assessed by Real-Time RT-PCR and protein expression and phosphorylation by Western-Blot. ( n = 4 − 6) Data = Mean ± SEM. P Results CNTF reduced DM1 incidence by 80% in WT but only by 10% in SOCS3kd mice. In WT, CNTF increased STAT3 and reduced STAT1 phosphorylation, while in SOCS3kd mice CNTF further increased STAT3 but failed to reduce STAT1. STZ reduced STAT3 and increased STAT1 phosphorylation, while in SOCS3kd mice STZ failed to reduce STAT3 but further increased STAT1. CNTF prevented STZ-induced pancreatic islets apoptosis and IL1beta-induced MIN6 cells apoptosis in WT mice and cells, but not in SOCS3kd or iNOSkd cells. The incidence of STZ-induced DM1 was lower in iNOSkd mice (20% of Control), and CNTF failed to further protect these mice. Nevertheless, CNTF effects over upstream pathways, such as increased STAT3 and SOCS3, and reduced STAT1, were similar to those of WT mice. In iNOSkd cells, CNTF partially prevented IL-1beta-induced cell-death, even though its upstream effects were similar to the WT cells, as we observed in vivo . Conclusion CNTF protects against STZ-induced DM1 and against cytokine-induced beta-cell death. These effects are dependent on the increase in SOCS3 expression. The cytokine-induced apoptosis as well as the pro-survival effect of CNTF over beta-cells depend on the balance between STAT1/STAT3 activation.

Protective effect of ciliary neurotrophic factor on retinal ganglion cell in acute ocular hypertension rat

Background Glaucoma associates with apoptosis of retinal ganglion cells ( RGCs).Research showed that ciliary neurotrophic factor (CNTF) can repair optic nerve trauma,but it has not reported whether CNTF has a protective effect on glaucomatous optic neuropathy. Objective This experimental study was to explore the protective effect of CNTF on RGCs in rat eyes with acute ocular hypertension. Methods Ocular acute hypertension models were induced in bilateral eyes of 24 clean Wistar rats by forced perfusion of a balanced salt solution into the anterior chamber.Two days before molding,0.5 μg recombinant human CNTF (5 μl) was injected intravitreously in the left eyes,and 5 mmol/L sodium phosphate solution (5 μl) was injected in the same way as the control group.Three other Wistar rats were used as the normal control group.The animals were sacrificed by excessive anesthesia and the retinal sections were prepared 1,3,7,14 days after molding.The morphology of the retina was examined and RGCs counting was performed by hematoxylin & eosin staining and observed under a light microscope.The expression of glutamic acid in RGCs was assessed by immunochemistry. Results Regular retinal structure with clearly defined cell layers were observed in normal control rats.Changes in vacuoles in RGCs were seen in the model control group.The number of degenerative RGCs decreased in the CNTF group.Compared with the model control group,number of normal RGCs increased from 1 day to 14 days after molding with a significant difference between the two groups (all P=0.000).Immunochemistry assay showed that the numbers of positive cells for glutamic acid were 5.50±1.04 and 6.00±1.41 for the average of 3 fields at 3 days or 7 days in the CNTF group,and those in the model control group were 9.00±2.91 and 10.83 ± 1.94,respectively,showing significant differences between them ( all P =0.000 ).However,no comparable differences were found in the numbers of positive cells for glutamic acid at 1 day and 14 days between the two groups( P=0.578,0.180). Conclusions CNTF down-regulates the expression of glutamic acid in RGCs and offers neuroprotection for RGCs in an acute glaucoma rat model. Key words: Ciliary neurotrophie factor; Ocular hypertention; Retinal ganglion cell; Glutamic acid

The Role of Nuclear Factor- B in Oxidative Stress Associated with Rabies Virus Infection (P03.263)

Objective: To investigate the role of NF-κB in rabies virus-induced oxidative stress in neurons. Background Recent studies in an experimental mouse model of rabies have shown that there are major structural changes in the brain involving neuronal processes that are associated with severe clinical disease. Cultured adult dorsal root ganglion (DRG) neurons are a good in vitro model for studying mechanisms involved in rabies virus – induced degeneration of neurites, because, unlike other neuronal cell types, these neurons are permissive to rabies virus infection. DRG neurons infected with the CVS strain of rabies virus show axonal swellings and immunostaining for 4-hydroxy-2-nonenal (4-HNE), indicating evidence of lipid peroxidation associated with oxidative stress, and also reduced axonal growth vs. mock-infected DRG neurons. Design/Methods: The expression and localization of NF-κB were evaluated by Western immunoblotting and immunofluorescence, respectively, in mock- versus CVS-infected rat DRG neurons. The effects of ciliary neurotrophic factor (CNTF), a potent activator of NF-κB, and SN50, a peptide inhibitor of NF-κB, were also evaluated. Results: CNTF had no effect on mock-infected rat DRG neurons, and was neuroprotective for CVS-neurons in reducing the number of 4-HNE-labeled puncta. SN50 and CVS infection had an additive effect in producing axonal swellings, suggesting that NF-κB is neuroprotective. CVS infection induced the expression of protein for the p50 subunit of NF-κB. The fluorescent signal for p50 was quantitatively evaluated in the nucleus and cytoplasm of mock- and CVS-infected rat DRG neurons. At 24 hrs p.i. there was a significant increase in the nucleus:cytoplasm ratio, indicating increased transcriptional activity of NF-kB, perhaps as a response to stress, whereas at both 48 and 72 hrs p.i. there was significantly reduced nuclear localization of NF-kB. Conclusions: CVS infection may induce oxidative stress by preventing nuclear activation of NF-κB. A rabies virus protein may directly inhibit NF-kB activity. Supported by: Canadian Institutes of Health Research. Disclosure: Dr. Jackson has nothing to disclose. Dr. Kammouni has nothing to disclose. Dr. Hasan has nothing to disclose. Dr. Fernyhough has nothing to disclose.