Recombinant Dog Ciliary neurotrophic factor receptor subunit alpha (CNTFR)

What is the molecular structure of canine CNTFR and how does it compare to other species?

Canine CNTFR shares a high degree of homology with human, mouse, and rat CNTFR at both nucleotide and amino acid levels. The canine CNTFR coding sequence is 1119 bp, encoding a protein of 372 amino acids (identical in length to human, mouse, and rat counterparts). Sequence analysis shows remarkable conservation across mammalian species:

The canine CNTFR amino acid sequence contains conserved hallmarks of cytokine receptors, including cysteine residue clusters, putative N-glycosylation sites, and the cytokine receptor consensus motif (WSXWS box). Interestingly, canine CNTFR is longer than chicken CNTFR (372 vs. 362 amino acids) .

How is CNTFR expressed in the canine retina, and what does this suggest about its function?

CNTFR shows a complex expression pattern in the canine retina, with both transcript and protein detected in multiple cell types. In situ hybridization and immunocytochemistry studies reveal that CNTFR is expressed in:

• Retinal pigment epithelium (RPE)

• Photoreceptors (both rods and cones)

• Inner nuclear layer (INL) cells

• Ganglion cells

In photoreceptors, CNTFR labeling is present at the level of the external limiting membrane (ELM) and in the proximal region of the inner segments (particularly the myoid). Expression is most intense in the central retina and decreases toward the periphery .

This widespread expression pattern, particularly in photoreceptors, suggests that CNTF could have a direct effect on these cells rather than acting indirectly through other retinal cells. This contrasts with findings in rodents, where CNTF's protective effects on photoreceptors are thought to be mediated indirectly through Müller cells or other INL cells .

What is the genomic location of the canine CNTFR gene and how does it relate to the human gene?

The canine CNTFR gene has been mapped to chromosome 11 (CFA 11) using radiation hybrid (RH) mapping with the RH083000 canine-hamster panel. Specifically, it is positioned approximately:

• 70.41 cR3000 telomeric to the microsatellite REN275MO5

• 56.19 cR3000 centromeric from the gene markers IFNA3/IFNA1

This chromosomal location exhibits conserved synteny with the cytogenetic p13 region of human chromosome 9 (HSA 9p13), which is the known location of the human CNTFR gene. This conservation of syntenic relationships across species indicates the evolutionary preservation of this genomic region and supports the orthologous relationship between the canine and human CNTFR genes .

What techniques are effective for detecting and characterizing canine CNTFR expression in tissues?

Multiple complementary techniques have proven effective for studying canine CNTFR:

RT-PCR Analysis:

• PCR amplification of a 369-bp product using primers CNTFR 6F and CNTFR 2R

• 30 amplification cycles at an annealing temperature of 58°C

• Products analyzed by electrophoresis on ethidium bromide–stained polyacrylamide gel (6%)

• Controls include DNase I digestion and omission of reverse transcriptase

In Situ Hybridization:

• Useful for cellular localization of CNTFR mRNA

• Requires both antisense (experimental) and sense (negative control) probes

• Provides detailed spatial information about transcript distribution

• Shows most intense labeling in central retina with decreased expression toward periphery

Immunohistochemistry/Immunocytochemistry:

• Successful detection using a rabbit protein A-purified polyclonal antibody raised against chicken CNTFR recombinant protein (1:2,000 dilution)

• This antibody shows cross-reactivity across mammalian species

• Can be performed on 7 μm thick cryosections from retinal tissues

• Allows visualization of protein expression at cellular and subcellular levels

Combined use of these techniques enables comprehensive characterization of CNTFR expression at both mRNA and protein levels, with consistent results confirming expression in multiple retinal cell types.

How can recombinant dog CNTFR be produced and purified for research applications?

While the search results don't specifically address dog CNTFR production, strategies can be adapted from human CNTFR production methodologies:

Expression Systems:

• Baculovirus-insect cell system: Preferred for glycosylated mammalian proteins requiring proper folding and post-translational modifications

• Mammalian expression systems (CHO, HEK293): Provide proper machinery for complex glycosylation

• E. coli expression systems: Less suitable if glycosylation is required for function

Protein Construction and Tags:

• Based on human CNTFR approaches, the amino acid sequence corresponding to Met1-Pro346 could be used

• C-terminal polyhistidine tag can facilitate purification

• Potential truncation to include only the extracellular domain for soluble CNTFR production

Purification Strategy:

• Immobilized metal affinity chromatography (IMAC) if His-tagged

• Additional purification via ion exchange chromatography

• Size exclusion chromatography for final polishing

• Rigorous quality control including endotoxin testing (<1.0 EU per μg) using LAL method

• Purity assessment by SDS-PAGE (>98% purity target)

Characterization:

• Functional validation through binding assays with CNTF

• Specific activity determination using ELISA-based binding assays

• Western blot verification using anti-CNTFR antibodies

• Mass spectrometry for accurate molecular weight and identification

What signaling mechanisms are activated downstream of CNTFR in canine cells?

CNTFR functions as part of a multi-subunit receptor complex. The complete signaling mechanism involves:

• Receptor Complex Formation:

  • Extracellular CNTF binding subunit (CNTFRα)

  • Two transmembrane signal transduction proteins:

    • Glycoprotein gp130

    • LIF receptor (LIFR)

• Signal Transduction:

  • While canine-specific pathways aren't explicitly described in the search results, based on homology with human and other mammalian systems, the following pathways are likely involved:

  • JAK/STAT pathway activation (particularly STAT3)

  • Potential activation of MAPK/ERK pathways

  • PI3K/Akt pathway involvement in survival signaling

• Soluble Receptor Dynamics:

  • CNTFR can exist as a membrane-bound receptor (through GPI linkage) or as a soluble form (sCNTFRα)

  • sCNTFRα can bind CNTF in solution, and this complex can act on cells expressing only LIFR and gp130 (without CNTFR)

  • This mechanism expands the range of cells that can respond to CNTF

• Cross-talk with Other Cytokine Pathways:

  • Due to shared use of gp130 with other cytokines (IL-6 family), potential for pathway cross-talk exists

  • This may explain some of the pleiotropic effects observed with CNTF

How might recombinant CNTFR be used to study retinal degeneration in canine disease models?

Recombinant CNTFR offers several valuable applications for studying retinal degeneration:

Research Applications in Canine Models:

• Receptor Expression Studies:

  • Comparison of CNTFR levels between normal and degenerating retinas

  • Evaluation of expression changes during disease progression in models like rcd1 and XLPRA2

• Signaling Pathway Analysis:

  • Investigation of downstream pathways activated by CNTF/CNTFR in canine retinal cells

  • Identification of potential disruptions in signaling in disease states

• Therapeutic Mechanism Investigation:

  • Determination whether CNTF acts directly on photoreceptors through CNTFR binding

  • Assessment of CNTFR-independent effects of CNTF to understand the complete mechanism of action

• Binding Competition Assays:

  • Use of soluble recombinant CNTFR to compete with endogenous receptor

  • Modulation of CNTF availability to specific cell populations

• Development of Improved Delivery Systems:

  • Creation of CNTF/CNTFR complexes that could have enhanced activity or specificity

  • Testing of sustained delivery systems for long-term treatment effects

The discovery that CNTFR is expressed in both rod and cone photoreceptors in the canine retina suggests that CNTF could have direct effects on these cells, potentially opening novel treatment pathways for retinal degenerations in dogs and, by extension, in humans .

What is the significance of canine CNTFR for CNTF-based therapeutic development?

The expression pattern of CNTFR in canine tissues has significant implications for therapeutic development:

Therapeutic Relevance:

• Direct vs. Indirect Action:

  • The expression of CNTFR in canine photoreceptors suggests CNTF could act directly on these cells

  • This contrasts with rodent studies suggesting indirect action through Müller cells or other INL cells

  • Species differences highlight the importance of studying CNTFR in models more closely related to humans

• Treatment Response Prediction:

  • CNTFR expression patterns might predict which cell populations will respond to CNTF treatment

  • Variable response to treatment in different retinal regions could correlate with CNTFR expression levels

• Clinical Trial Design Implications:

  • Understanding CNTFR expression in disease states could inform:

    • Patient selection strategies

    • Optimal timing of interventions

    • Appropriate dosing regimens

    • Predictive biomarkers for response

• Translational Relevance:

  • Dogs with inherited retinal degenerations serve as important large animal models

  • Dog breeds with naturally occurring retinal diseases that mimic human conditions offer advantages over induced rodent models

  • Findings in canine studies may translate more directly to human applications

Despite these promising aspects, search result reports that in XLPRA2 dogs, intravitreal CNTF injections failed to prevent photoreceptor death in central and mid-peripheral retina, highlighting the complexity of developing effective treatments.

How does CNTFR expression compare between normal and diseased canine retinas?

The available search results provide limited direct comparative data between normal and diseased retinas, but they offer some insights:

CNTFR Expression in Normal vs. Diseased States:

• Developmental Expression:

  • CNTFRα is expressed in normal photoreceptors during postnatal retinal maturation (4-24 weeks of age)

  • This expression appears to be maintained in the adult canine retina

• Disease Model Expression:

  • CNTFRα expression has been studied in mutant photoreceptors during the course of retinal degeneration

  • Specifically examined in rcd1 and XLPRA2 dog models

  • The presence of CNTFR in these models suggests expression is maintained during disease progression

• Spatial Distribution:

  • In normal retinas, CNTFR expression is most intense in the central retina and decreases toward the periphery

  • This spatial gradient may have implications for regional differences in disease progression or treatment response

• Therapeutic Response Correlation:

  • Despite CNTFR expression in XLPRA2 dogs, CNTF treatment failed to prevent photoreceptor death in central and mid-peripheral retina

  • This suggests that CNTFR presence alone may not be sufficient for therapeutic efficacy

  • Other factors in the signaling pathway or cellular environment might influence the response to CNTF

A comprehensive comparative analysis of CNTFR expression patterns between normal and various disease states would be valuable for better understanding the potential utility of CNTF-based therapies.

What functional assays can be used to evaluate recombinant canine CNTFR activity?

While the search results don't specifically describe assays for canine CNTFR, several approaches can be adapted from those used with human CNTFR:

Binding Assays:

• ELISA-Based Binding Assessment:

  • Immobilize recombinant CNTFR at an optimal concentration (e.g., 10 μg/ml)

  • Test binding to biotinylated CNTF across a concentration range (e.g., 1.28-160 ng/ml)

  • Establish binding affinity and specificity parameters

• Surface Plasmon Resonance (SPR):

  • Real-time measurement of CNTF-CNTFR binding kinetics

  • Determination of association and dissociation rate constants

  • Calculation of binding affinity (KD)

Cellular Functional Assays:

• Cell Proliferation Assays:

  • Based on the observation that CNTF stimulates proliferation of TF-1 cells

  • This effect can be enhanced up to 100-fold by the addition of recombinant CNTFR

  • Measurement of ED50 values for proliferative response

• Signal Transduction Analysis:

  • Assessment of downstream pathway activation (e.g., STAT3 phosphorylation)

  • Western blotting for phosphorylated signaling molecules after CNTF stimulation

  • Comparison of signaling in the presence/absence of recombinant CNTFR

Complex Formation Assays:

• Analysis of Receptor Complex Assembly:

  • Study of CNTFR interaction with gp130 and LIFR

  • Co-immunoprecipitation experiments to detect complex formation

  • Investigation of how CNTFR/CNTF complex interacts with cells expressing only gp130/LIFR

Biological Response Assays:

• Neurite Outgrowth Assays:

  • Assessment of CNTF-induced neurite extension in neuronal cultures

  • Quantification of neurite length, branching, and growth cone characteristics

  • Evaluation of how recombinant CNTFR affects these parameters

What are the challenges in developing specific antibodies for canine CNTFR?

Developing specific antibodies for canine CNTFR presents several technical challenges:

Antibody Development Challenges:

• Cross-Reactivity Considerations:

  • While canine CNTFR shares high homology with human/mouse/rat proteins, epitope differences may exist

  • Antibodies developed against one species may have variable cross-reactivity with canine CNTFR

  • Studies have successfully used antibodies raised against chicken CNTFR for canine detection, despite lower homology

• Recombinant Protein Availability:

  • Access to well-characterized recombinant canine CNTFR is essential for antibody development

  • Search result notes: "Because the source of the recombinant chick CNTFR used to block the anti-chick CNTFR antibody is no longer available, we could not repeat these experiments..."

  • This highlights how reagent availability can impact research progress

• Validation Requirements:

  • Thorough validation is needed to ensure specificity for CNTFR versus related receptors

  • Multiple techniques (Western blotting, immunoprecipitation, immunohistochemistry) should confirm consistent results

  • Ideally, validation would include tissues from CNTFR knockout models or siRNA knockdown experiments

• Conformational Epitopes:

  • CNTFR's complex structure with multiple domains may present conformation-dependent epitopes

  • Different antibodies may be needed for detecting native versus denatured forms

  • Post-translational modifications like glycosylation may affect epitope recognition

• Applications Across Techniques:

  • Antibodies suitable for one application (e.g., Western blotting) may not work for others (e.g., immunohistochemistry)

  • Polyclonal antibodies offer broader epitope recognition but potentially more cross-reactivity

  • Monoclonal antibodies provide specificity but might recognize limited epitopes

Current research has successfully used a rabbit protein A-purified polyclonal antibody raised against chicken CNTFR recombinant protein at 1:2,000 dilution for canine studies .

How do the structural features of canine CNTFR influence its function in neuronal survival?

The structural features of canine CNTFR play crucial roles in determining its function:

Structure-Function Relationships:

• Receptor Domains and Ligand Binding:

  • Canine CNTFR contains characteristic cytokine receptor features:

    • Clusters of cysteine residues (important for structural integrity)

    • Putative N-glycosylation sites (affecting protein stability and function)

    • Cytokine receptor consensus motif (WSXWS box) critical for proper folding and ligand binding

  • These conserved elements suggest similar binding mechanisms across species

• Membrane Attachment:

  • Like human CNTFR, canine CNTFR is likely anchored to cell membranes by a glycosylphosphatidylinositol (GPI) linkage

  • This GPI linkage allows for release of soluble CNTFR by phospholipase action

  • The soluble form can bind CNTF and activate cells expressing only gp130 and LIFR

• Receptor Complex Formation:

  • CNTFR associates with gp130 and LIFR to form a complete signaling complex

  • This tripartite receptor complex activates intracellular signaling cascades

  • The organization of this complex is crucial for proper signal transduction

• Neuronal Survival Mechanisms:

  • CNTF binding to CNTFR leads to activation of pathways that promote neuronal survival

  • In the retina, CNTFR expression in photoreceptors suggests direct survival effects on these cells

  • This contrasts with the indirect mechanism proposed in rodents

  • The high conservation of CNTFR structure across species "suggests that the CNTFR signaling pathway is conserved across evolution"

• Species-Specific Considerations:

  • Despite high conservation, the lower homology between dog and chicken CNTFR compared to other mammals might affect:

    • Ligand binding affinity

    • Signaling efficiency

    • Receptor complex stability

  • These subtle differences could influence therapeutic responses across species

What are the most promising applications of recombinant dog CNTFR for neurodegenerative disease research?

Recombinant dog CNTFR shows significant potential for advancing neurodegenerative disease research:

Research Applications:

• Development of Novel Therapeutic Biologics:

  • Creation of CNTFR/CNTF complexes with enhanced stability or target specificity

  • Engineering of modified CNTFR variants with improved pharmacokinetic properties

  • Development of soluble CNTFR as a potential therapeutic agent to expand CNTF responsiveness to cells lacking membrane-bound CNTFR

• Retinal Degeneration Models:

  • Use in naturally occurring canine models of retinal degeneration (rcd1, XLPRA2)

  • Investigation of why CNTF failed to prevent photoreceptor death despite CNTFR expression

  • Exploration of combined approaches targeting multiple pathways

• Comparative Studies Across Species:

  • Detailed comparison of CNTFR signaling between canine and human systems

  • Better understanding of species-specific differences that might affect translational research

  • Development of canine-specific tools to overcome limitations of cross-species reagent use

• Mechanistic Studies of CNTF Action:

  • Investigation of direct versus indirect mechanisms of CNTF neuroprotection

  • Elucidation of the complete signaling complex and downstream pathways in canine cells

  • Understanding why the expression of CNTFR doesn't always correlate with therapeutic response

• Broader Neurodegenerative Applications:

  • Extension beyond retinal studies to other neurodegenerative conditions

  • Investigation of potential roles in inflammatory neurological conditions

  • Exploration of CNTFR's role in multiple sclerosis models, based on CNTF's effects in experimental autoimmune encephalomyelitis

How might genetic manipulation of CNTFR expression influence retinal degeneration progression?

Genetic manipulation of CNTFR offers intriguing possibilities for understanding and treating retinal degeneration:

Genetic Modification Approaches:

• Overexpression Studies:

  • Increased CNTFR expression might enhance responsiveness to endogenous or therapeutic CNTF

  • Could potentially compensate for decreasing CNTFR levels during disease progression

  • Viral vector-mediated delivery could target specific retinal cell populations

• Knockdown/Knockout Approaches:

  • Reduced CNTFR expression could help determine if CNTF's effects are exclusively mediated through CNTFR

  • Would clarify if alternative pathways exist for CNTF signaling

  • Could reveal if CNTFR has ligand-independent functions

• Cell-Type Specific Manipulation:

  • Selective modification of CNTFR in photoreceptors versus Müller cells or RPE

  • Would help resolve the debate about direct versus indirect mechanisms of CNTF action

  • Could optimize therapeutic approaches by targeting the most relevant cell populations

• Mutation Correction:

  • For inherited retinal degenerations with CNTFR variants, gene editing could restore normal function

  • This could determine whether CNTFR dysfunction contributes to disease pathogenesis

• Potential Outcomes:

  • Enhanced CNTFR expression might improve therapeutic responses to CNTF

  • Better understanding of cell-specific roles in disease progression

  • Development of combined approaches targeting CNTFR and other pathways

The observation that CNTFR is expressed in photoreceptors but CNTF treatment failed in some models suggests complex interactions that genetic manipulation studies could help unravel .

What comparative analyses between human and canine CNTFR would advance translational research?

Comparative analyses between human and canine CNTFR would significantly enhance translational research efforts:

Comparative Research Priorities:

• Detailed Structural Comparison:

  • Crystal structure determination of both human and canine CNTFR

  • Binding site analysis to identify subtle differences that might affect ligand interactions

  • Investigation of how the 93% sequence identity translates to functional similarities/differences

• Expression Pattern Comparison:

  • Comprehensive mapping of CNTFR expression across tissues in both species

  • Comparison of retinal expression patterns, particularly in photoreceptors

  • Analysis of whether disease-related expression changes are consistent across species

• Signaling Pathway Analysis:

  • Side-by-side comparison of CNTF-induced signaling pathways

  • Investigation of potential species-specific downstream effectors

  • Determination if differences exist in signal transduction efficiency or duration

• Therapeutic Response Comparison:

  • Examination of whether human and canine cells respond similarly to CNTF

  • Analysis of potential differences in dose-response relationships

  • Determination if therapeutic failures in canine models predict human outcomes

• Cross-Reactivity Studies:

  • Development of reagents that interact equivalently with both species' receptors

  • Assessment of whether humanized biologics targeting CNTFR pathway work in canine models

  • Creation of biologics that could be used across species for better translational research

Such comparative analyses would strengthen the value of canine models in developing therapies for human retinal degeneration and other neurological disorders, potentially reducing translational failures and accelerating therapeutic development .