Ciliary Neurotrophic Factor (CNTF) Inhibits In Vitro Cementoblast Mineralization and Induces Autophagy, in Part by STAT3/ERK Commitment.

In animal models, the administration of ciliary neurotrophic factor (CNTF) was demonstrated to reduce bone mass and to participate in bone remodeling. Cementoblasts, a cell type embedded in the cementum, are the main cells to produce and mineralize the extracellular matrix. The effect of CNTF on cementoblasts has not yet been addressed. Thus, the goal of this in vitro study was to investigate possible influences of exogenous CNTF on cementogenesis, as well as autophagy regulation and subsequent mechanisms in cementoblasts. Cementoblasts (OCCM-30) were stimulated with exogenous CNTF. Alizarin Red staining was performed to analyze the functional differentiation (mineralization) of OCCM-30 cells. The release of OPG was quantified by ELISA. The expression of cementogenesis markers (RUNX-2, OCN, BMP-7, BSP, and SPON-2) was evaluated by RT-qPCR. Western blotting (WB) was performed for the protein expression of STAT3, COX-2, SHP-2, cPLAα, cPLAß; ERK1/2, P38, and JNK. The autophagic flux was assessed using WB and RT-qPCR analysis of LC3A/B, Beclin-1, and Atg-5, and the autophagosome was investigated by immunofluorescence staining (IF). The ERK1/2 (FR180204) or STAT3 (sc-202818) antagonist was added, and the cellular response was analyzed using flow cytometry. Exogenous CNTF significantly attenuated mineralized nodule formation, impaired OPG release, and downregulated the mRNA levels of RUNX-2, OCN, BMP-7, and BSP. Moreover, CNTF induced the phosphorylation of STAT3 and activated a transient activation of SHP-2, cPLAß, ERK1/2, P38, and JNK protein. CNTF also induced autophagosome formation and promoted autophagy-associated gene and protein expressions. Additionally, the inhibition of ERK1/2 or STAT3 reversed a CNTF-induced mineralization impairment and had regulatory effects on CNTF-induced autophagosome formation. Our data revealed that CNTF acts as a potent inhibitor of cementogenesis, and it can trigger autophagy, in part by ERK1/2 and STAT3 commitment in the cementoblasts. Thus, it may play an important role in inducing or facilitating inflammatory root resorption during orthodontic tooth movement.

Polyethylene glycol-coupled IGF1 delays motor function defects in a mouse model of spinal muscular atrophy with respiratory distress type 1.

Spinal muscular atrophy with respiratory distress type 1 is a neuromuscular disorder characterized by progressive weakness and atrophy of the diaphragm and skeletal muscles, leading to death in childhood. No effective treatment is available. The neuromuscular degeneration (Nmd(2J)) mouse shares a crucial mutation in the immunoglobulin mu-binding protein 2 gene (Ighmbp2) with spinal muscular atrophy with respiratory distress type 1 patients and also displays some basic features of the human disease. This model serves as a promising tool in understanding the complex mechanisms of the disease and in exploring novel treatment modalities such as insulin-like growth factor 1 (IGF1) which supports myogenic and neurogenic survival and stimulates differentiation during development. Here we investigated the treatment effects with polyethylene glycol-coupled IGF1 and its mechanisms of action in neurons and muscles. Polyethylene glycol-coupled IGF1 was applied subcutaneously every second day from post-natal Day 14 to post-natal Day 42 and the outcome was assessed by morphology, electromyography, and molecular studies. We found reduced IGF1 serum levels in Nmd(2J) mice 2 weeks after birth, which was normalized by polyethylene glycol-coupled IGF1 treatment. Nmd(2J) mice showed marked neurogenic muscle fibre atrophy in the gastrocnemius muscle and polyethylene glycol-coupled IGF1 treatment resulted in muscle fibre hypertrophy and slowed fibre degeneration along with significantly higher numbers of functionally active axonal sprouts. In the diaphragm with predominant myogenic changes a profound protection from muscle fibre degeneration was observed under treatment. No effects of polyethylene glycol-coupled IGF1 were monitored at the level of motor neuron survival. The beneficial effects of polyethylene glycol-coupled IGF1 corresponded to a marked activation of the IGF1 receptor, resulting in enhanced phosphorylation of Akt (protein kinase B) and the ribosomal protein S6 kinase in striated muscles and spinal cord from Nmd(2J) mice. Based on these findings, polyethylene glycol-coupled IGF1 may hold promise as a candidate for future treatment trials in human patients with spinal muscular atrophy with respiratory distress type 1.

Efficacy of PEGylated ciliary neurotrophic factor superagonist variant in diet-induced obesity mice.

Ciliary neurotrophic factor (CNTF) is a neurotrophic cytokine able to induce appetite reduction, weight loss and antidiabetic effects. However, its susceptibility to neutralizing anti-CNTF antibodies in patients hampered its use for treatment of human obesity and diabetes. In addition, CNTF has a very short plasma half-life, which limits its use as a therapeutic agent. Solutions, directed to prolong its in vivo effects, vary from the implantation of encapsulated secreting cells to identification of more active variants or chemical modification of the protein itself. PEGylation is a widely used modification for shielding proteins from circulating antibodies and for increasing their plasma half-life. Here, we have selected DH-CNTF, a CNTF variant which has a 40-fold higher affinity for the CNTF receptor α accompanied by an increased activity in cellular assays. The PEGylated DH-CNTF retained the biological activity of native protein in vitro and showed a significant improvement of pharmacokinetic parameters. In an acute model of glucose tolerance, the PEG-DH-CNTF was able to reduce the glycemia in diet-induced obese animals, with a performance equaled by a 10-fold higher dose of DH-CNTF. In addition, the PEGylated DH-CNTF analog demonstrated a more potent weight loss effect than the unmodified protein, opening to the use of CNTF as weight reducing agent with treatment regimens that can better meet patient compliance thanks to reduced dosing schedules.

Impaired expression of ciliary neurotrophic factor in Charcot-Marie-Tooth type 1A neuropathy.

We investigated the contribution of Schwann cell-derived ciliary neurotrophic factor (CNTF) to the pathogenesis of Charcot-Marie-Tooth disease type 1A (CMT1A) and addressed the question as to whether it plays a role in the development of axonal damage observed in the disease, with aging. Ciliary neurotrophic factor was underexpressed in experimental CMT1A but not in other models of hereditary neuropathies. Sciatic nerve crush experiments and dosage of CNTF at different time points showed that expression of this trophic factor remained significantly lower in CMT1A rats than in normal controls; moreover, in uninjured CMT1A sciatic nerves CNTF levels further decreased with ageing, thus paralleling the molecular signs of axonal impairment, that is increased expression of non-phosphorylated neurofilaments and amyloid precursor protein. Administration of CNTF to dorsal root ganglia cultures reduced dephosphorylation of neurofilaments in CMT1A cultures, without improving demyelination. Taken together, these results provide further evidence that the production of CNTF by Schwann cells is markedly reduced in CMT1A. Moreover, the observations suggest that trophic support to the axon is impaired in CMT1A and that further studies on the therapeutic use of trophic factors or their derivatives in experimental and human CMT1A are warranted.

Recombinant ciliary neurotrophic factor promotes nerve regeneration and induces gene expression in silicon tube-bridged transected sciatic nerves in adult rats.

Sciatic nerves in adult male rats were transected and reunited via a silicone chamber. This was followed by a focal injection of recombinant ciliary neurotrophic factor (CNTF). To evaluate the effect of this therapeutic approach and to explore its possible mechanisms, nerve regeneration was traced by horseradish peroxidase retrograde labeling. Functional recovery was evaluated by functional assessment of the hind feet and the expression of a number of proteins was detected using immunohistochemistry. The results showed that a single administration of CNTF could promote regeneration of motor axons, with improved functional recovery in adult rats. Growth associated protein (GAP)-43, S100, CD68 and major histocompatibility complex class II immunoreactivity in the regenerative and distal nerves suggested that CNTF could promote axon regeneration, Schwann cell migration, monocyte infiltration and activation. CNTF might also indirectly promote axonal regeneration by further activating the JAK-STAT3 pathway and subsequently upregulating phosphotyrosine, GAP-43 and S100 expression to enhance proliferation, growth and migration of Schwann cells. CNTF has suggested important targets for pharmacological intervention in peripheral nerve disease and injury.

Development of long-acting ciliary neurotrophic factor by site-specific conjugation with different-sized polyethylene glycols and transferrin.

To overcome the deficiency of rapid elimination from blood, the truncated human recombinant ciliary neurotrophic factor was formulated by site-specific attachment of different-sized PEG-maleimide or by cross-linking with human transferrin through a hetero-bi-functional PEG linker (NHS-PEG5k-MAL). The PEGylated CNTF was purified by a two-step chromatography procedure and the transferrin coupling CNTF conjugate was separated through an elegant protocol. The conjugation site on CNTF was identified by peptide mapping analysis and validated that the linkage of the conjugates was specifically happened to Cys17 residue. Although both PEGylated and transferrin coupling CNTF demonstrated decreased cell based residual activity, markedly enhanced pharmacokinetic behaviors in normal male Sprague-Dawley rats were observed, especially for the PEG40k-CNTF with approximately 58-times improvement compared with the unmodified counterpart. The evaluation of the in vivo potency of body weight-losing was performed with normal male C57BL6 mice and the results revealed that both PEGylation and transferrin coupling could achieve improved therapeutic benefits relative to that of CNTF. Besides, PEG20k/40k-CNTF demonstrated more effective than transferrin coupling CNTF (Tf-PEG5k-CNTF) despite that the later showed preferable pharmacokinetic profile and cell based residual activity compared with PEG20k-CNTF. Weekly subcutaneous administration of PEG40k-CNTF with 0.5mg/kg and 1.0mg/kg dose resulted in approximately 35% and 50% decrease in food intake during one interval period of injection, indicating that PEG40k-CNTF is the most potential anti-obese agent for therapeutics.

Stimulation of neurite outgrowth by neurotrophins delivered from degradable hydrogels.

Degradable hydrogels are useful vehicles for the delivery of growth factors to promote the regeneration of diseased or damaged tissue. In the central nervous system, there are many instances where the delivery of neurotrophins has great potential in tissue repair, especially for treatment of spinal cord injury. In this work, hydrogels based on poly(ethylene glycol) that form via a photoinitiated polymerization were investigated for the delivery of neurotrophins. The release kinetics of these factors are controlled by changes in the network crosslinking density, which influences neurotrophin diffusion and subsequent release from the gels with total release times ranging from weeks to several months. The release and activity of one neurotrophic factor, ciliary-neurotrophic factor (CNTF), was assessed with a cell-based proliferation assay and an assay for neurite outgrowth from retinal explants. CNTF released from a degradable hydrogel above an explanted retina was able to stimulate outgrowth of a significantly higher number of neurites than controls without CNTF. Finally, unique microsphere/hydrogel composites were developed to simultaneously deliver multiple neurotrophins with individual release rates.

Dose-dependent neurochemical and functional protection afforded by encapsulated CNTF-producing cells.

Intracerebral delivery of hCNTF has shown considerable neuroprotective potential in animal models of Huntington's disease (HD). The present study describes the relationship between a range of hCNTF doses and the resulting behavioral and neurochemical (striatal ChAT and GAD activity) protection in a rodent model of HD. Encapsulated BHK delivering a range of hCNTF doses were implanted into the lateral ventricle ipsilateral to an intrastriatal quinolinic acid (QA) injection. Results demonstrated a dose-dependent effect of hCNTF with complete, partial, and no observable neuroprotection occurring with preimplant doses of hCNTF of 30.8, 8.6, and 0.8-2.1 ng hCNTF/24 h, respectively. These data continue to support the use of cellular delivery of hCNTF for HD and will facilitate the optimization of this approach in the clinical situation.

The regrowth of axons within tissue defects in the CNS is promoted by implanted hydrogel matrices that contain BDNF and CNTF producing fibroblasts.

In this study we demonstrate the potential for combining biocompatible polymers with genetically engineered cells to elicit axon regrowth across tissue defects in the injured CNS. Eighteen- to 21-day-old rats received implants of poly N-(2-hydroxypropyl)-methacrylamide (HPMA) hydrogels containing RGD peptide sequences that had been infiltrated with control (untransfected) fibroblasts (n = 8), fibroblasts engineered to express brain-derived neurotrophic factor (BDNF) (n = 5), ciliary neurotrophic factor (CNTF) (n = 5), or a mixture of BDNF and CNTF expressing fibroblasts (n = 11). Fibroblasts were prelabeled with Hoechst 33342. Cell/polymer constructs were inserted into cavities made in the left optic tract, between thalamus and superior colliculus. After 4-8 weeks, retinal projections were analyzed by injecting right eyes with cholera toxin (B-subunit). Rats were perfused 24 h later and sections were immunoreacted to visualize retinal axons, other axons (RT97 antibody), host astrocytes and macrophages, donor fibroblasts, and extracellular matrix molecules. The volume fraction (VF) of each gel that was occupied by RT97(+) axons was quantified. RT-PCR confirmed expression of the transgenes prior to, and 5 weeks after, transplantation. Compared to control rats (mean VF = 0.02 +/- 0.01% SEM) there was increased ingrowth of RT97(+) axons into implants in CNTF (mean VF = 0.33 +/- 0.19%) and BDNF (mean VF = 0.62 +/-0.19%) groups. Axon growth into hydrogels in the mixed BDNF/CNTF group (mean VF = 3.58 +/- 0.92%) was significantly greater (P < 0.05) than in the BDNF or CNTF fibroblast groups. Retinal axons exhibited a complex branching pattern within gels containing BDNF or BDNF/CNTF fibroblasts; however, they regrew the greatest distances within implants containing both BDNF and CNTF expressing cells.