Recombinant Human Ciliary neurotrophic factor protein (CNTF) (Active)

What is the basic structural and functional characterization of Recombinant Human CNTF?

Recombinant Human CNTF is a full-length protein spanning amino acids 2-200, typically expressed in HEK 293 cells for research applications. The protein has a predicted molecular weight of 22856.99 Da (±10 Da by ESI-TOF), with observed mass spectrometry values of approximately 22858.40 Da . The active form maintains ≥95% purity with endotoxin levels ≤0.005 EU/μg, making it suitable for sensitive experimental applications including cell culture and functional studies .

CNTF functions primarily as a survival factor for various neuronal cell types, with particularly documented efficacy in preventing degeneration of motor axons following axotomy . It belongs to the CNTF cytokine family and shares structural similarities with IL-6, suggesting overlapping yet distinct signaling mechanisms .

How does CNTF signal transduction operate in neuronal and glial cells?

CNTF signaling involves a multicomponent receptor complex including the CNTF receptor α (CNTFRα), which can be expressed on both neuronal and glial cell populations. Unlike related cytokines such as IL-6 that activate STAT-3 and ERK phosphorylation pathways, CNTF exhibits a distinct signaling profile . Research demonstrates that CNTF induces specific patterns of protein phosphorylation and dephosphorylation, including phosphorylation of LYN substrate-1 and β-tubulin 5 .

The signaling efficacy of CNTF can be significantly enhanced through addition of exogenous soluble CNTFRα (sCNTFRα), which facilitates CNTF's ability to collaborate with other factors such as IFNγ to modulate cellular responses . This complex signaling has important methodological implications for experimental design, as researchers must consider both direct effects on neurons and indirect effects mediated through glial intermediaries.

What are the validated biological assays for confirming CNTF activity?

The biological activity of Recombinant Human CNTF is typically determined through dose-dependent proliferation assays using TF-1 cells. The effective dose (ED50) for biological activity is ≤4.914 μg/ml, corresponding to a specific activity of approximately 2.04 × 10^5 units/mg . This standardized assay provides a reliable method for quality control and activity confirmation.

Alternative validation approaches include:

• Measurement of neuropeptide expression changes (e.g., urocortin-1 mRNA upregulation)

• Assessment of microglial CD40 surface expression when CNTF is combined with sCNTFRα and IFNγ

• Evaluation of metabolic alterations in target tissues, particularly shifts toward aerobic glycolysis

Each method provides distinct insights into CNTF functionality and should be selected based on specific research questions.

What are the optimal storage and handling conditions for maintaining CNTF bioactivity?

For maintaining maximum bioactivity of Recombinant Human CNTF, researchers should follow these evidence-based protocols:

• Storage temperature: Store lyophilized CNTF at -20°C and reconstituted protein at -80°C

• Reconstitution: Use sterile, buffer-controlled solutions (PBS or equivalent) for reconstitution

• Aliquoting: Prepare single-use aliquots immediately after reconstitution to avoid freeze-thaw cycles

• Working concentrations: Effective concentrations typically range from 1-50 ng/ml for in vitro studies

• Stability considerations: Reconstituted protein maintains >90% activity for up to 2 weeks at 4°C

Importantly, as CNTF is an active protein capable of eliciting biological responses in vivo, all preparations should be handled with appropriate caution to prevent unintended exposure or cross-contamination .

How should researchers design appropriate controls for CNTF experiments?

Robust experimental design for CNTF research requires multiple control conditions:

• Vehicle controls: Identical buffer composition without CNTF protein

• Concentration gradients: Multiple CNTF concentrations to establish dose-response relationships

• Time-course analyses: Assessment at multiple timepoints (e.g., 24h, 48h, 72h) as CNTF-induced effects may show temporal dynamics

• Receptor blocking: Anti-CNTFRα antibodies or receptor antagonists to confirm specificity

• Signaling pathway inhibitors: Selective inhibitors of downstream pathways to elucidate mechanisms

For in vivo experiments, controls should include both vehicle treatments and biologically inactive CNTF variants to distinguish between specific and non-specific effects.

What analytical techniques are most effective for measuring CNTF-induced changes in protein phosphorylation?

Based on current research methodologies, the following analytical approaches are recommended for investigating CNTF-induced phosphorylation changes:

Researchers have successfully employed 2-D western blot analysis to demonstrate that CNTF induces dephosphorylation of one set of proteins while simultaneously promoting phosphorylation of others, including LYN substrate-1 and β-tubulin 5 . This technique allows for visualization of global phosphorylation landscapes when combined with phospho-specific staining methods.

How does CNTF demonstrate neuroprotective effects in retinal degeneration models?

CNTF has shown significant neuroprotective properties in retinal degeneration models through multiple mechanisms:

• Metabolic modulation: CNTF treatment alters the metabolic status of degenerating retinas, specifically by elevating aerobic glycolysis and enhancing anabolism . This metabolic reprogramming appears to be a fundamental mechanism underlying CNTF's neuroprotective effects.

• Structural preservation: In clinical trials for retinitis pigmentosa, CNTF treatment resulted in increased retinal thickness, suggesting a preservation of retinal structure even in progressive disease conditions .

• Functional improvement: Recent clinical trials for macular telangiectasia type 2 have demonstrated that CNTF treatment leads to both morphological and visual function improvements across multiple assessment parameters .

When designing experiments to evaluate CNTF's neuroprotective effects, researchers should incorporate both structural and functional endpoints, along with metabolic analyses to fully characterize the protective mechanisms.

What methodological approaches are most effective for studying CNTF effects on neuronal-glial interactions?

To effectively investigate CNTF effects on neuronal-glial interactions, researchers should consider these methodological approaches:

• Co-culture systems: Establish neuron-glia co-cultures with selective depletion of specific glial populations to dissect differential responses.

• Conditioned media experiments: Collect media from CNTF-treated glial cells and apply to neuronal cultures to identify secreted factors mediating indirect effects.

• Cell-specific receptor knockdown: Use siRNA or CRISPR techniques to selectively inhibit CNTF receptor components in either neurons or glia.

• In vivo cell-specific reporters: Employ transgenic models with cell-type specific reporters to monitor activation patterns following CNTF administration.

• Microdialysis coupled with proteomics: Apply in vivo microdialysis with proteomic analysis to identify secreted factors in response to CNTF treatment.

Research has demonstrated that CNTF stimulates astrocytes to secrete FGF-2 and microglia to secrete glial cell line-derived neurotrophic factor (GDNF), suggesting that CNTF can promote motor neuron survival indirectly through glial intermediaries . These interactions highlight the importance of considering both direct and indirect effects when evaluating CNTF's neuroprotective mechanisms.

What are the critical parameters for intracerebroventricular (i.c.v.) administration of CNTF in animal models?

For researchers conducting i.c.v. administration of CNTF in animal models, the following parameters have been validated:

• Surgical coordinates: For mouse models, placement of a 26-gauge stainless steel guide cannula should use flat-skull coordinates from bregma: anteroposterior -0.825 mm, mediolateral 0 mm, dorsoventral -4.8 mm .

• Recovery period: Allow 7-14 days of recovery following cannula implantation, with daily handling for at least one week prior to experimental manipulations .

• Administration timing: For feeding studies, CNTF administration 1 hour prior to the onset of the dark phase has shown efficacy .

• Dosage: 1 μg CNTF in 2 μl total volume (0.5 mg/ml) delivered by slow infusion over 10-15 minutes through a 30-gauge needle has demonstrated significant biological effects .

• Control groups: Include 0.9% saline vehicle controls administered through identical protocols.

• Endpoint timing: For c-Fos activation studies, tissue collection at 2 hours post-administration has shown optimal detection of neuronal activation patterns .

This protocol has demonstrated efficacy in reducing food intake and body weight in ad libitum-fed wild type mice, with effects lasting at least 2 hours post-administration .

How can researchers leverage CNTF's effects on energy homeostasis for metabolic disease investigations?

CNTF demonstrates significant effects on energy homeostasis pathways that can be leveraged for metabolic disease research through several approaches:

• Hypothalamic neuropeptide regulation: CNTF regulates expression of key neuropeptides involved in energy homeostasis, particularly increasing urocortin-1 mRNA expression by 1.84-fold at 48 hours post-treatment . Researchers can exploit this by:

  • Measuring changes in multiple neuropeptide levels simultaneously (urocortin-1, urocortin-2, AgRP, BDNF, neurotensin)

  • Conducting time-course analyses to capture both acute and chronic adaptations

  • Using targeted knockdown approaches to determine the necessity of specific neuropeptides for CNTF's metabolic effects

• Metabolomic profiling: CNTF treatment enhances aerobic glycolysis and anabolism in tissues , suggesting researchers should incorporate comprehensive metabolomic analyses to fully characterize these shifts.

• Integration with obesity models: As CNTF reduces feeding and induces weight loss , researchers can investigate its therapeutic potential in various obesity models while monitoring both central and peripheral metabolic adaptations.

These approaches can provide deeper insights into how neurotrophic factors might be leveraged to address metabolic disorders through central nervous system mechanisms.

What considerations should guide the design of experiments investigating CNTF effects on microglial phenotypes?

When investigating CNTF effects on microglial phenotypes, researchers should consider:

• Receptor expression: Murine microglia express CNTF receptor α (CNTFRα), which can be induced by interferon-γ (IFNγ) . Baseline and stimulated receptor expression should be quantified.

• Soluble receptor supplementation: CNTF responses in microglia can be enhanced by adding exogenous soluble CNTFRα (sCNTFRα) . Experimental designs should include conditions with and without sCNTFRα supplementation.

• Cytokine interactions: CNTF collaborates with IFNγ to increase microglial CD40 surface expression, with pronounced effects when microglia are differentiated toward dendritic-like cells . This suggests the importance of examining CNTF effects in various microglial activation states.

• Phosphoprotein profiling: Unlike IL-6, CNTF does not activate STAT-3 and ERK phosphorylation in microglia, indicating distinct signaling mechanisms . Comprehensive phosphoprotein analysis should be incorporated.

• Functional outputs: Beyond phenotypic markers, researchers should assess functional outcomes including:

  • Secreted factors (cytokines, neurotrophins)

  • Phagocytic capacity

  • Migration and chemotaxis

  • Neurotoxicity/neuroprotection in co-culture systems

These considerations will help researchers develop more nuanced experimental approaches to understanding CNTF's immunomodulatory effects in the central nervous system.

What approaches can resolve contradictions in the literature regarding CNTF signaling mechanisms?

To address contradictions in CNTF signaling literature, researchers should consider:

• Cell type specificity: CNTF signaling differs substantially between neuronal and glial populations. Experiments should include multiple cell types under identical conditions to document differential responses.

• Receptor complex heterogeneity: The CNTF receptor complex involves multiple components that may vary between tissues. Comprehensive characterization of receptor expression patterns should precede signaling studies.

• Temporal dynamics: Short-term versus long-term CNTF exposure may activate different signaling cascades. Time-course experiments with multiple endpoints are essential.

• Species differences: Human and rodent CNTF may exhibit subtle but important functional differences. Where possible, cross-species comparisons should be included.

• Technical variations: Different detection methods for signaling intermediates may yield conflicting results. Multiple complementary techniques should be employed to verify key findings.

A systematic approach incorporating these considerations can help reconcile apparently contradictory findings and develop a more unified understanding of CNTF signaling mechanisms.

What methodologies have been validated for evaluating CNTF efficacy in retinal degeneration clinical trials?

Clinical trials evaluating CNTF for retinal degenerative diseases have utilized several validated methodologies:

• Encapsulated cell technology: Trials have employed implants containing genetically engineered cells that produce a secreted form of recombinant human CNTF . This delivery system provides sustained local release while avoiding systemic exposure.

• Retinal thickness measurements: Optical coherence tomography (OCT) has proven effective for quantifying changes in retinal thickness following CNTF treatment, with trials for retinitis pigmentosa demonstrating increased thickness in treated patients .

• Visual function assessment: Multiple parameters beyond best-corrected visual acuity have shown utility, particularly in macular telangiectasia type 2 trials, where morphological and functional improvements have been documented using complementary assessment methods .

• Metabolic analysis: Evaluation of metabolic shifts, particularly toward enhanced aerobic glycolysis and anabolism, provides mechanistic insights into CNTF's neuroprotective effects .

Researchers designing translational studies should incorporate these validated approaches while considering disease-specific modifications to detection methods and outcome measures.

How can researchers distinguish direct versus indirect effects of CNTF on neuronal survival?

To distinguish direct versus indirect effects of CNTF on neuronal survival, researchers should implement:

These approaches can help resolve the complex interplay between direct neuronal effects and those mediated through glial intermediaries, providing a more complete understanding of CNTF's neuroprotective mechanisms.