Recombinant Mouse Ninjurin-2 (Ninj2)

What is the molecular structure and basic characteristics of mouse Ninjurin-2?

Ninjurin-2 (nerve injury-induced protein 2) is a 20-22 kDa transmembrane protein belonging to the Ninjurin family of cell adhesion molecules. Mouse Ninjurin-2 shares approximately 71% amino acid identity with human Ninjurin-2 over amino acids 1-65 . The protein has an unusual membrane orientation featuring:

• A 65 amino acid N-terminal extracellular domain (ECD) that mediates homophilic binding

• First transmembrane segment

• Cytoplasmic region

• Second transmembrane segment

• C-terminal extracellular domain (approximately amino acids 128-142)

Unlike many other cell adhesion molecules, Ninjurin-2 does not share adhesion motifs with its family member Ninjurin-1, although they do share conserved hydrophobic regions in their transmembrane domains .

What are the primary expression patterns of Ninjurin-2 in mouse tissues?

In the peripheral nervous system, Ninjurin-2 expression follows tissue-specific patterns:

Unlike Ninjurin-1, Ninjurin-2 expression dramatically elevates in differentiated postmitotic neurons during development, suggesting distinct regulatory mechanisms and functions between these family members .

How does Ninjurin-2 function as a cell adhesion molecule?

Ninjurin-2 functions primarily as a homophilic adhesion molecule, meaning it binds to other Ninjurin-2 molecules on adjacent cells. Research demonstrates:

• Aggregation assays with Jurkat cells stably transfected with Ninjurin-2 show significantly increased cellular aggregation (83±4%) compared to control cells (14±4%) .

• The N-terminal extracellular domain mediates this adhesion, with specific peptides derived from this region capable of inhibiting Ninjurin-2-mediated cellular aggregation.

• Unlike Ninjurin-1, Ninjurin-2 does not share comparable adhesion motifs, and the two proteins do not interact with each other despite their structural similarities .

• This homophilic binding capability supports neurite outgrowth from DRG neurons via Ninjurin-2-mediated cellular interactions .

This adhesive function is particularly relevant for neuronal development and regeneration processes after injury.

How does Ninjurin-2 regulate myelination in the peripheral nervous system, and what methodological approaches best demonstrate this function?

Ninjurin-2 acts as a negative regulator of myelination in the peripheral nervous system through interaction with integrin signaling pathways. This finding has been demonstrated through several key methodological approaches:

• Genetic manipulation in mouse models:

  • SC-specific Ninjurin-2 knockout mice (Dhh^cre/+;Ninj2^fl/fl^) exhibit precocious myelination in sciatic nerves

  • Higher numbers of myelinated axons and smaller bundle areas are observable at P0-P3 in knockout mice compared to wild-type

  • Similar phenotypes observed in Cnp^cre/+;Ninj2^fl/fl^ mice, confirming the result in another glial-specific knockout strain

• Ultrastructural analysis:

  • Transmission electron microscopy revealed accelerated radial sorting in the sciatic nerves of knockout mice

  • No change in G-ratio, suggesting normal myelin thickness despite accelerated timing

  • Myelinogenesis becomes comparable by P7 between wild-type and knockout mice

• Functional assessment approaches:

  • CatWalk gait analysis to assess functional outcomes

  • Electrophysiological recordings of compound muscle action potentials (CMAPs)

  • Measurement of nerve conduction velocity and CMAP amplitude

• Cellular proliferation analysis:

  • Immunofluorescent imaging of Sox10+ (SC marker), Mpz+ (mature SC marker), Ki67+ (proliferation marker), and TUNEL+ (apoptosis marker) cells

  • Quantification of cellular populations and proliferation rates

The results demonstrate that Ninjurin-2 deletion specifically promotes Schwann cell proliferation without affecting cell survival, leading to an enlarged SC population and accelerated myelination .

What is the relationship between Ninjurin-2 and p53, and how does this interaction impact cellular function?

Ninjurin-2 and p53 form a complex feedback regulatory loop with significant implications for cellular growth and tumor biology:

• NINJ2 as a p53 target:

  • NINJ2 can be induced by p53 following DNA damage (demonstrated in MCF7 and Molt4 cells treated with doxorubicin or camptothecin)

  • Chromatin immunoprecipitation (ChIP) assays revealed p53 binding to the NINJ2 promoter, which increases following doxorubicin treatment

  • A p53-responsive element (p53-RE) has been identified in the NINJ2 promoter region

• NINJ2 as a regulator of p53:

  • NINJ2 can modulate p53 expression by repressing both wild-type and mutant p53 mRNA translation

  • Loss of NINJ2 leads to increased p53 protein levels and induction of p53 targets like p21

  • This regulation occurs at the translational level (demonstrated using Click-iT metabolic labeling of newly synthesized p53 protein)

• Differential effects based on p53 status:

  • Loss of NINJ2 inhibits cell growth in wild-type p53-containing cells

  • Loss of NINJ2 promotes cell growth in mutant p53-containing cells

  • This creates a context-dependent role for NINJ2 in cellular growth regulation

This bidirectional relationship has profound implications for understanding cancer biology and potential therapeutic approaches targeting this pathway.

What molecular mechanisms mediate Ninjurin-2's effects on Schwann cell development, and how can these be experimentally manipulated?

Ninjurin-2 regulates Schwann cell development primarily through interference with laminin-integrin signaling. This mechanism has been elucidated through several experimental approaches:

• RNA-Seq analysis of wild-type and Ninjurin-2 knockdown Schwann cells revealed:

  • 1,163 upregulated genes and 879 downregulated genes following Ninjurin-2 knockdown

  • Gene ontology analysis showed enrichment for biological processes including myelination, ensheathment of neurons, glial cell development, and integrin-mediated signaling pathways

  • KEGG pathway analysis identified ECM-receptor interaction, focal adhesion, and cell adhesion molecules as significantly affected pathways

• Protein interaction studies:

  • Co-immunoprecipitation and mass spectrometry identified ITGB1 (Integrin β1) as a major Ninjurin-2 interacting protein

  • This interaction was confirmed through reciprocal co-immunoprecipitation assays

  • Ninjurin-2 was found to interrupt the binding of laminin (LN211) to ITGB1

• Signaling pathway analysis:

  • Loss of Ninjurin-2 activated downstream FAK signaling (part of laminin-integrin signaling)

  • Overexpression of Ninjurin-2 abolished LN211-induced FAK activation

  • This established Ninjurin-2 as a negative regulator of the laminin-integrin-FAK signaling axis

• Experimental manipulation:

  • Administration of GRGDSP, an integrin inhibitor, blocked FAK activation in Ninjurin-2-deficient cells

  • GRGDSP treatment significantly delayed the accelerated remyelination process in Ninjurin-2 knockout mice after sciatic nerve injury

  • This pharmacological intervention confirmed the mechanistic role of integrin signaling in mediating Ninjurin-2's effects

These findings establish a clear molecular mechanism whereby Ninjurin-2 negatively regulates Schwann cell development by interfering with laminin-integrin signaling through direct interaction with ITGB1.

How can Ninjurin-2's role in colorectal cancer growth be exploited for potential therapeutic applications?

Ninjurin-2 has been identified as a potential therapeutic target in colorectal cancer (CRC) based on its overexpression and functional role in promoting cancer cell growth through several mechanisms:

• Expression analysis in cancer tissues:

  • NINJ2 is overexpressed in established CRC cell lines (HT-29) and primary CRC cells

  • NINJ2 shows elevated expression in human colon cancer tissues compared to normal colon tissues and epithelial cells

• Functional characterization methodologies:

  • Loss-of-function approaches:

    • NINJ2 shRNA silencing significantly inhibits CRC cell survival and proliferation

    • CRISPR/Cas9-mediated NINJ2 knockout (achieving >95% reduction in protein levels) similarly reduces cell growth

    • Both approaches induce significant apoptosis in CRC cells

  • Gain-of-function approaches:

    • Lentivirus-mediated NINJ2 overexpression promotes CRC cell proliferation

• Mechanistic investigation:

  • Co-immunoprecipitation studies revealed NINJ2 interactions with multiple receptor tyrosine kinases (RTKs), including:

    • Epidermal growth factor receptor (EGFR)

    • Platelet-derived growth factor receptors α/β (PDGFRα/β)

    • Fibroblast growth factor receptor (FGFR)

  • NINJ2 modulates downstream RTK signaling:

    • NINJ2 silencing/knockout inhibits Akt and Erk activation

    • NINJ2 overexpression augments Akt and Erk signaling pathways

• In vivo validation:

  • NINJ2-silenced or NINJ2-knockout CRC xenografts grow significantly slower than control xenografts in SCID mice

  • Decreased p-Akt and p-Erk1/2 levels in NINJ2-depleted tumor tissues confirm the mechanism operates in vivo

  • Daily tumor growth calculations showed dramatic inhibition of growth rates with NINJ2 depletion

These findings suggest several potential therapeutic approaches that could be developed:

• Direct NINJ2 inhibitors that disrupt its interaction with RTKs

• Combination therapies targeting both NINJ2 and downstream Akt/Erk pathways

• Stratification of patients based on NINJ2 expression levels for personalized therapy approaches

What are the optimal methods for detecting and quantifying mouse Ninjurin-2 in experimental systems?

Multiple complementary approaches can be employed for detecting and quantifying Ninjurin-2, each with specific advantages:

When selecting antibodies, consider:

• Species cross-reactivity (human/mouse/rat)

• Epitope location (N-terminal vs. C-terminal)

• Detection sensitivity in different applications

• Background in specific tissues

For recombinant protein production, the N-terminal extracellular domain (aa 1-65) is often used as it contains the functional adhesion region and can be efficiently produced in E. coli expression systems .

What experimental design considerations are important when studying Ninjurin-2's role in peripheral nerve injury and regeneration?

Investigating Ninjurin-2's role in peripheral nerve injury and regeneration requires careful experimental design:

• Animal model selection:

  • Sciatic nerve crush injury model provides a standardized approach for studying remyelination

  • Consider timing of injury in relation to developmental stage (adult mice at P60 are commonly used)

  • Include appropriate Ninjurin-2 knockout models (Dhh^cre/+;Ninj2^fl/fl^ for Schwann cell-specific deletion)

  • Include time-matched controls for each analysis timepoint

• Time course analysis:

  • Multiple post-injury timepoints are essential (typical schedule: 0, 7, 14, 21, 28, 35 days post-injury)

  • Each timepoint requires separate groups of animals

  • Consider both early (demyelination/proliferation) and late (remyelination) phases

• Multi-level assessment approach:

  • Functional analysis:

    • Gait analysis using DigiGait system

    • Electrophysiological measurements (CMAPs, nerve conduction velocity)

  • Molecular analysis:

    • Expression profiling of myelin markers (Mpz)

    • Cell-specific markers (Sox10 for Schwann cells)

    • Proliferation markers (Ki67)

  • Structural analysis:

    • Immunofluorescence for cell counting and marker co-localization

    • Electron microscopy for myelin ultrastructure

    • Quantification of myelinated axon numbers and bundle area

• Pharmacological intervention:

  • Integrin inhibitor GRGDSP can be used to inhibit laminin-integrin signaling

  • Administration schedule and dose must be optimized (typically administered at early stages of remyelination)

  • Vehicle controls must be included

  • Consider both systemic and local delivery methods

• Statistical considerations:

  • Minimum of 6 mice per experimental group is recommended

  • Student's t-test for comparing two groups

  • Report exact P-values with significance thresholds (*P < 0.05, **P < 0.01, ***P < 0.001)

  • Present data with appropriate error bars (typically mean ± SEM)

How can contradictory findings about Ninjurin-2's role in central vs. peripheral nervous system myelination be reconciled experimentally?

Research has revealed that Ninjurin-2 exerts opposite effects on myelination in the central versus peripheral nervous systems. This apparent contradiction requires careful experimental approaches to reconcile:

• Comparative experimental design:

  • Parallel analysis of CNS oligodendrocytes and PNS Schwann cells in the same genetic models

  • Use of cell-type specific conditional knockouts:

    • Dhh^cre/+;Ninj2^fl/fl^ for Schwann cell-specific deletion

    • Cnp^cre/+;Ninj2^fl/fl^ or oligodendrocyte-specific Cre drivers for CNS studies

  • Consistent assessment metrics across both systems

• Protein interaction analysis:

  • Comprehensive interactome profiling in both cell types:

    • Ninjurin-2 predominantly interacts with ITGB1 in Schwann cells

    • Ninjurin-2 interacts with TNFR1 in oligodendrocytes

  • Validation of differential binding partners through reciprocal co-immunoprecipitation

  • Functional validation of these interactions using pathway inhibitors

• Signaling pathway comparison:

  • Examination of laminin/integrin signaling components in both cell types

  • Assessment of TNFα/TNFR1 pathway activation in both systems

  • Phosphorylation status of downstream effectors (e.g., FAK for integrin pathway)

• Reconciliation strategies:

  • Cell-specific expression of binding partners may determine Ninjurin-2 function

  • Subcellular localization studies may reveal different compartmentalization

  • Assessment of developmental timing effects on protein expression and interaction

  • Investigation of potential post-translational modifications that might differ between cell types

This systematic approach can help explain why loss of Ninjurin-2 promotes myelination in the PNS but inhibits oligodendrocyte and myelin development in the CNS, revealing important context-dependent functions of this protein.

What experimental approaches are most effective for studying the Ninjurin-2/p53 feedback loop in different cell contexts?

The complex feedback loop between Ninjurin-2 and p53 requires sophisticated experimental approaches to fully characterize:

• Genetic manipulation systems:

  • Isogenic cell line pairs with NINJ2 knockout (CRISPR/Cas9) in both wild-type and p53-mutant backgrounds

  • Compound mouse models (Ninj2^-/-;p53^-/-) compared to single knockouts

  • Inducible expression systems for temporal control of either protein

• Transcriptional regulation analysis:

  • ChIP assays to detect p53 binding to the NINJ2 promoter under various conditions:

    • Basal state

    • Following DNA damage (doxorubicin, camptothecin)

    • Different p53 mutant variants

  • Reporter assays with wild-type and mutated p53-response elements from NINJ2 promoter

• Translational regulation assessment:

  • Click-iT metabolic labeling to track newly synthesized p53 protein

  • Polysome profiling to assess p53 mRNA translation efficiency

  • RNA immunoprecipitation to detect potential NINJ2 interactions with translation machinery

• Functional outcome measurement:

  Cell Context	NINJ2 Loss	NINJ2 Overexpression	Key Assays
  Wild-type p53 cells	Growth inhibition	Growth promotion	Colony formation, cell proliferation
  Mutant p53 cells	Growth promotion	Growth inhibition	Colony formation, tumor sphere formation
  p53-null cells	Minimal effect	Minimal effect	Comparative growth assays

• Mechanistic dissection:

  • RNA-seq and proteomics in different genetic backgrounds to identify context-dependent targets

  • Domain mapping to identify regions of NINJ2 critical for p53 regulation

  • Assessment of post-translational modifications of both proteins

  • Drug sensitivity profiling in different genetic contexts

This integrated approach can help delineate the complex bidirectional relationship between NINJ2 and p53, explaining the context-dependent functions observed in different experimental systems.

What are the most promising directions for developing Ninjurin-2-targeted therapies based on current research findings?

Several promising therapeutic strategies emerge from current Ninjurin-2 research:

• Cancer therapy approaches:

  • Development of small molecule inhibitors targeting the Ninjurin-2/RTK interface

  • Therapeutic antibodies against the N-terminal extracellular domain

  • Combination therapies targeting both Ninjurin-2 and downstream Akt/Erk pathways

  • p53 status-dependent intervention strategies

• Nerve regeneration enhancement:

  • Transient inhibition of Ninjurin-2 to accelerate remyelination after peripheral nerve injury

  • Development of peptide inhibitors based on the integrin-binding region

  • Targeted delivery systems to specifically modulate Ninjurin-2 in Schwann cells

  • Temporal control of inhibition to match regeneration phases

• Stroke therapeutics:

  • Investigation of NINJ2 polymorphisms linked to ischemic stroke for personalized medicine

  • Modulation of endothelial Ninjurin-2 to reduce TLR4-mediated inflammation

  • Development of biomarkers based on Ninjurin-2 expression or polymorphic variants

• Technical considerations for therapeutic development:

  • The unique membrane topology of Ninjurin-2 provides multiple potential target sites

  • The N-terminal extracellular domain (aa 1-65) represents the most accessible target region

  • Cell-type specific delivery strategies will be important given differential functions in CNS vs PNS

  • Consideration of potential compensatory mechanisms involving Ninjurin-1