RTN4R Antibody

What is RTN4R and why is it important in neuroscience research?

RTN4R (Reticulon 4 Receptor), also known as Nogo receptor or NgR, is a cell membrane protein expressed primarily in the central nervous system, with highest expression levels in the gray matter of the brain. In humans, the canonical protein has 473 amino acid residues with a molecular mass of approximately 50.7 kDa . RTN4R plays a crucial role in regulating axonal regeneration and plasticity in the central nervous system through the activation of rho kinase .

The importance of RTN4R in neuroscience stems from its role as a receptor for multiple myelin-associated inhibitory factors including RTN4 (also called Nogo), oligodendrocyte myelin glycoprotein (OMG), and myelin-associated glycoprotein (MAG) . When activated, RTN4R initiates signaling cascades that inhibit axonal growth and restrict synaptic plasticity, which has significant implications for neural development, injury response, and neurological disorders.

What are the known structural features and post-translational modifications of RTN4R?

RTN4R contains a leucine-rich repeat domain that is critical for ligand binding, as revealed in the 1.65 Å crystal structure of the BAI1/RTN4-receptor complex . The protein undergoes several important post-translational modifications including:

• O-glycosylation: Modification of serine or threonine residues with glycans

• N-glycosylation: Addition of glycans to asparagine residues

These modifications are not merely decorative but functional - the crystal structure of the BAI1/RTN4-receptor complex shows that C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by these unusual glycoconjugates, enabling high-affinity interactions .

What specific applications are RTN4R antibodies most commonly used for?

Based on the research literature, RTN4R antibodies are primarily utilized in the following applications:

Western blotting is particularly valuable for detecting RTN4R expression levels and validating antibody specificity, while immunohistochemistry and immunofluorescence provide spatial information about RTN4R distribution in tissue sections or cultured cells .

How should I select the appropriate RTN4R antibody for my specific experimental needs?

When selecting an RTN4R antibody, consider these critical factors:

Species reactivity: Ensure the antibody recognizes RTN4R from your model organism. Many RTN4R antibodies show cross-reactivity across human, mouse, and rat samples, but confirmation is essential . RTN4R gene orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species .

Antibody type: Decide between polyclonal and monoclonal antibodies based on your needs:

• Polyclonal antibodies recognize multiple epitopes and offer higher sensitivity but potentially lower specificity

• Monoclonal antibodies target a single epitope and provide higher specificity and reproducibility

Application compatibility: Verify that the antibody has been validated for your specific application. Some antibodies work well for Western blotting but not for immunohistochemistry or vice versa .

Epitope location: For studying specific RTN4R domains or variants, select antibodies targeting relevant epitopes. For instance, if studying the R292H variant associated with schizophrenia, an antibody recognizing this region would be preferable .

Validation evidence: Review literature citations and validation data provided by suppliers to ensure reliability for your experimental system .

What are the optimal conditions for using RTN4R antibodies in Western blotting?

For optimal Western blot results with RTN4R antibodies, follow these methodological guidelines:

Sample preparation:

• Extract proteins from tissues or cells using a lysis buffer containing protease inhibitors

• For brain tissue samples, use region-specific extraction to account for differential expression (gray matter shows highest RTN4R expression)

• Include DTT or β-mercaptoethanol in your sample buffer as RTN4R may form oligomers

Gel electrophoresis:

• Use 10-12% SDS-PAGE gels for optimal separation of RTN4R (50.7 kDa)

• Include positive controls such as brain tissue lysates

Transfer and blocking:

• Transfer to PVDF or nitrocellulose membranes

• Block with 5% non-fat milk or BSA in TBS-T

Antibody incubation:

• Primary antibody: Dilute RTN4R antibodies typically between 1:100-1:500 in blocking buffer

• Secondary antibody: Use appropriate species-specific HRP-conjugated antibodies

Detection:

• Expected band size: approximately 51 kDa for full-length RTN4R

• Potential additional bands may represent post-translationally modified forms or alternatively spliced variants

What controls should be included when performing immunostaining with RTN4R antibodies?

When performing immunostaining experiments with RTN4R antibodies, include these critical controls:

Positive tissue controls:

• Brain tissue sections, particularly cerebral cortex (gray matter), which shows high RTN4R expression

• Cell lines with known RTN4R expression

Negative controls:

• Primary antibody omission: Incubate sections with secondary antibody only

• Isotype control: Use non-specific IgG from the same species as the primary antibody

• Peptide competition: Pre-absorb the antibody with the immunizing peptide (if available) to confirm specificity

Blocking peptide validation:
For peptide competition assays, reconstitute the blocking peptide in distilled water (typically 200μl to obtain a 0.5mg/ml solution). Mix equal volumes of peptide and antibody at the required dilution and incubate at ambient temperature for approximately 20 minutes. Run two identical blots in parallel - one with antibody alone and one with antibody pre-adsorbed to the peptide for direct comparison .

How can RTN4R antibodies be used to investigate neural regeneration and psychiatric disorders?

RTN4R antibodies can be powerful tools for studying both neural regeneration mechanisms and psychiatric disorders:

Neural regeneration studies:

• Use RTN4R antibodies to map expression patterns in various injury models

• Combine with functional assays to assess growth cone collapse, as demonstrated in studies of the R292H variant

• Employ RTN4R antibodies in co-immunoprecipitation experiments to identify novel interaction partners in the regeneration inhibitory pathway

• Visualize RTN4R distribution at injury sites using immunofluorescence with confocal microscopy

Psychiatric disorder investigations:

• RTN4R gene variants, particularly R292H, have been significantly associated with schizophrenia (p=0.048)

• Use RTN4R antibodies to compare expression levels in post-mortem brain tissue from patients with schizophrenia or autism spectrum disorders versus controls

• Investigate RTN4R expression in induced pluripotent stem cell (iPSC)-derived neurons from patients with 22q11.2 deletion syndrome, which is associated with a 20-30% prevalence of schizophrenia

• Study co-localization with other schizophrenia risk genes using multi-label immunofluorescence

In both contexts, RTN4R antibodies can help elucidate whether protein levels, subcellular localization, or post-translational modifications are altered in disease states or following injury.

What approaches can be used to study the interaction between RTN4R and its binding partners?

Several methodological approaches can be employed to investigate RTN4R interactions with binding partners such as RTN4, OMG, MAG, and BAI proteins:

Co-immunoprecipitation (Co-IP):

• Use RTN4R antibodies to pull down the receptor complex

• Western blot for potential binding partners

• Consider crosslinking to stabilize transient interactions

Proximity Ligation Assay (PLA):

• Allows visualization of protein interactions in situ with single-molecule resolution

• Requires antibodies against both RTN4R and its potential partners from different species

Surface Plasmon Resonance (SPR):

• Quantitative analysis of binding kinetics

• Has been used successfully to measure nanomolar affinities between RTN4Rs and BAIs

• The BAI3-RTN4R interaction showed the highest affinity (Kd = 1.9 nM), while the BAI1-RTN4RL2 interaction displayed the lowest affinity at 30.0 nM

Cell adhesion assays:

• HEK 293F cells expressing RTN4R and binding partners can be used to study aggregation

• This approach demonstrated that BAI1- and BAI3-expressing cells strongly and specifically aggregate with cells expressing RTN4Rs, and that this interaction is calcium-independent

Structural studies:
The crystal structure of the BAI1/RTN4-receptor complex at 1.65 Å resolution revealed that unique post-translational modifications of the BAI TSR domain, including C-mannosylation of tryptophan and O-fucosylation of threonine, are critical for the interaction interface .

How can RTN4R antibodies be optimized for studying its role in dendritic arborization and synapse formation?

To study RTN4R's role in dendritic arborization and synapse formation, consider these specialized approaches:

Subcellular fractionation and localization:

• Use RTN4R antibodies in combination with subcellular fractionation to determine receptor distribution in dendrites, axons, and synaptic compartments

• Western blotting of purified synaptosomal preparations can reveal enrichment in specific compartments

High-resolution imaging:

• Super-resolution microscopy techniques (STED, STORM, PALM) with RTN4R antibodies can provide nanoscale localization

• Combine with markers for pre- and post-synaptic structures to study synaptic localization

Human iPSC-derived neuron cultures:

• RTN4R antibodies have been used to study RTN4R function in human neurons, where they regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial vs. neuronal BAIs

• Time-course experiments can reveal developmental regulation of RTN4R expression

Functional readouts:

• Combine with electrophysiological measurements to correlate RTN4R localization with synaptic function

• Use RTN4R antibodies for acute function-blocking experiments to assess immediate effects on synaptic transmission

What are common issues when using RTN4R antibodies and how can they be resolved?

How should RTN4R antibody results be interpreted in the context of known RTN4R variants and mutations?

When interpreting results from RTN4R antibody experiments, consider these variant-specific factors:

Known RTN4R variants:
Through mutation screening of RTN4R coding exons, researchers have discovered four rare (minor allele frequency <1%) missense mutations: R68H, D259N, R292H, and V363M . The antibody's epitope location determines whether these variants will be detected equivalently.

Interpretation guidelines:

• Verify whether the antibody's epitope overlaps with known mutation sites

• For Western blotting, mutations may not significantly alter apparent molecular weight but could affect antibody affinity

• In samples with heterozygous mutations, quantification may underestimate total RTN4R if the antibody has reduced affinity for the mutant form

• When studying patient samples with known RTN4R variants, consider using multiple antibodies targeting different epitopes for confirmation

R292H variant considerations:
This variant has been significantly associated with schizophrenia and affects growth cone formation . When studying this variant:

How do post-translational modifications affect RTN4R antibody recognition?

Post-translational modifications (PTMs) of RTN4R can significantly impact antibody recognition and experimental results:

N-glycosylation and O-glycosylation effects:

• RTN4R undergoes both N- and O-glycosylation, which can mask epitopes recognized by certain antibodies

• These modifications can shift apparent molecular weight in Western blots

• To assess the impact of glycosylation:

  • Treat samples with PNGase F (for N-glycans) or O-glycosidase (for O-glycans)

  • Compare treated and untreated samples via Western blot

  • Observe potential shifts in band patterns or intensity

Functional significance of PTMs:
The crystal structure of the BAI1/RTN4R complex revealed that C-mannosylation of tryptophan and O-fucosylation of threonine in BAI TSR-domains creates an interface with RTN4R that enables high-affinity interactions . These findings suggest that studying PTMs is not merely technical but biologically relevant to RTN4R function.

Recommendations:

• Use multiple antibodies targeting different epitopes to ensure comprehensive detection

• When comparing RTN4R levels across different tissues or conditions, consider potential variations in PTM patterns

• For studies focused on protein interactions, evaluate whether the antibody might disrupt or enhance binding interfaces where PTMs play a critical role

What emerging techniques could enhance RTN4R antibody-based research?

Several cutting-edge approaches show promise for advancing RTN4R research:

CRISPR-based tagging:

• Endogenous tagging of RTN4R with small epitope tags or fluorescent proteins

• Allows visualization of physiological RTN4R levels without antibody limitations

• Can be combined with proximity labeling approaches like BioID or APEX to map the RTN4R interactome

Single-cell proteomics:

• Analysis of RTN4R expression heterogeneity across brain cell populations

• Could reveal previously unappreciated expression patterns in rare neural subtypes

Spatial transcriptomics combined with antibody validation:

• Correlation of RTN4R mRNA expression patterns with protein localization

• Validation of antibody specificity through correlation with transcript abundance

Nanobody development:

• Smaller antibody fragments that may access epitopes inaccessible to conventional antibodies

• Potential for improved penetration in tissue sections and live-cell imaging

How might RTN4R antibodies contribute to therapeutic development for neurodegenerative conditions?

RTN4R antibodies could advance therapeutic strategies for neurodegenerative conditions in several ways:

Target validation:

• Use RTN4R antibodies to confirm target engagement in preclinical models

• Quantify RTN4R expression levels in affected tissues and correlate with disease progression

Function-blocking applications:

• Function-blocking RTN4R antibodies might promote axonal regeneration by interrupting interactions with inhibitory ligands

• These could be developed as therapeutic candidates for spinal cord injury or other CNS trauma

Biomarker development:

• RTN4R antibodies could be used to develop assays for soluble RTN4R fragments in CSF or blood

• Potential correlation with disease progression in neurodegenerative conditions

Delivery of therapeutic payloads:

• RTN4R-targeting antibodies could be used to deliver drugs specifically to neurons

• This approach might improve the therapeutic index of neuroprotective compounds