Recombinant Human Neurotrophin-4 protein (NTF4) (Active)

What is the basic structure of human Neurotrophin-4 protein?

Human Neurotrophin-4 (NT-4) is a member of the NGF family of neuronal and epithelial growth factors. The protein is synthesized as a 210 amino acid (aa) precursor comprising:

• A 24 aa signal sequence

• A 56 aa propeptide

• A 130 aa mature protein (spanning residues Gly81-Ala210)

The structural hallmark of NT-4, like all neurotrophins, is the characteristic arrangement of six conserved cysteine residues that form three disulfide bonds, known as the cysteine knot. This structure has also been found in other growth factors such as PDGF . Mature human NT-4 shares 48-52% amino acid sequence identity with human β-NGF, BDNF, and NT-3 .

How does recombinant NT-4 compare with endogenous NT-4 in terms of functionality?

Recombinant NT-4 retains the key functional properties of endogenous NT-4 when properly produced and folded. Most commercially available recombinant human NT-4 is produced in insect cell lines like Spodoptera frugiperda (Sf21) using baculovirus expression systems . The functionality of recombinant NT-4 can be verified through:

• TrkB receptor activation assays - Proper recombinant NT-4 should induce receptor dimerization and activation of TrkB

• ERK1/2 MAPK phosphorylation - As demonstrated in TrkB-transfected HEK-293 cells, where NT-4 induces concentration-dependent ERK1/2 activation that can be blocked by K252a (TrkB inhibitor)

• Neuronal survival assays - Effective concentration for promoting neuronal survival typically ranges from 0.3-3 ng/mL

When using recombinant NT-4, the biological activity should be validated through these functional assays rather than assumed based solely on protein concentration.

What distinguishes NT-4 from other neurotrophins in the same family?

NT-4 has several unique characteristics that distinguish it from other neurotrophins:

• Receptor specificity: NT-4 binds primarily to TrkB (shared with BDNF) and to p75NTR (which binds all neurotrophins)

• Expression pattern: NT-4 is expressed at highest levels in prostate, with lower levels in thymus, placenta, and skeletal muscle . It is also expressed in neurons of the superior cervical, stellate and celiac ganglion

• Sequence homology: While maintaining the conserved neurotrophin structure, NT-4 shares only 48-52% amino acid identity with other human neurotrophins

• Evolutionary conservation: Human NT-4 shares 91% and 95% amino acid sequence identity with mouse and rat NT-4/5, respectively, indicating strong evolutionary conservation

• Unique functions: Despite activating the same TrkB receptor as BDNF, NT-4 has distinct physiological roles due to differences in expression patterns and possibly signaling dynamics

What are the optimal storage and reconstitution conditions for recombinant NT-4?

For optimal stability and activity of recombinant NT-4, follow these guidelines:

Storage:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Store lyophilized protein at -20°C to -70°C for up to 12 months from receipt date

• After reconstitution, store at 2-8°C for up to 1 month under sterile conditions

• For longer storage after reconstitution, aliquot and store at -20°C to -70°C for up to 6 months

Reconstitution:

• For standard preparations (with carrier protein): Reconstitute at 50 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• For carrier-free preparations: Reconstitute at 50 μg/mL in sterile PBS

• For working solutions below 50 μg/mL, BSA (0.1 mg/ml) should be added to prevent protein adsorption to tubes and loss of activity

• Always centrifuge all protein preparations before use (10,000 × g for 5 minutes)

• Note that repeated freezing/thawing cycles may result in loss of activity

What detection methods are most reliable for studying NT-4 expression in tissues and cells?

Several complementary methods can be used to detect NT-4 with different advantages:

Immunoassays (ELISA):

• Sandwich immunoassays using specific antibodies like biotinylated anti-human NT-4 (e.g., BAF268) allow quantitative detection

• This approach offers high sensitivity and specificity for protein-level expression

In situ hybridization:

• Using DIG-labeled RNA probes prepared from human NT-4 cDNA fragments

• Example protocol: Use primers targeting nucleotide positions 374-839 from the first ATG of NT-4

• Visualization with alkaline phosphatase-conjugated anti-DIG (1:2,500) and NBT/BCIP color development (5.5 hr, 30°C)

• This method allows spatial localization of NT-4 mRNA in tissue sections

Western blot:

• For detecting NT-4 protein expression in cell lysates

• Use phosphorylation-specific antibodies when studying signaling pathways activated by NT-4 (e.g., anti-phospho-ERK1/2 for downstream signaling)

RT-PCR:

• For NT-4 mRNA detection

• Primer design targeting conserved regions is critical for specificity

• Example: Primers like NTF4-SF (5′-CTGCAGCTGGCGGCAGTCC-3′) and NTF4-SR (5′-ATTACCCTCAAGTTGCTCCA-3′) can generate a 466 bp fragment

How can I assess the biological activity of recombinant NT-4 in my experimental system?

To verify the biological activity of recombinant NT-4:

• TrkB receptor phosphorylation assay:

  • Use TrkB-transfected cell lines (e.g., HEK-293 cells)

  • Serum-starve cells for 2 hours

  • Pre-incubate with or without TrkB inhibitor (e.g., K252a at 200 nM)

  • Stimulate with various concentrations of NT-4

  • Assess phosphorylation of TrkB or downstream molecules like ERK1/2 by Western blot

• Neuronal survival assay:

  • Culture primary neurons (e.g., mesencephalic dopamine neurons)

  • Treat with NT-4 (effective concentration typically 0.3-3 ng/mL)

  • Assess survival using viability assays or morphological assessment

  • Include positive controls (e.g., BDNF) and negative controls

• Neurite outgrowth measurement:

  • NT-4 promotes dendritic outgrowth in cultured neurons

  • Quantify neurite length, branching complexity, and growth cone morphology

  • Compare to vehicle control and positive controls

• Calcium current measurements:

  • NT-4 has been shown to promote calcium currents in cultured mesencephalic dopamine neurons

  • Use electrophysiological techniques to measure changes in calcium currents after NT-4 treatment

What is known about NT-4's dual role in breast cancer progression?

Recent research has revealed that NT-4 plays a complex dual role in breast cancer:

Pro-metastatic effects:

• NT-4 promotes epithelial-mesenchymal transition (EMT), cell motility, and invasiveness of breast cancer cells in vitro and in vivo

• Mechanistically, NT-4 activates PRKDC/AKT and ANXA1/NF-κB pathways to stabilize SNAIL protein

• This leads to decreased E-cadherin levels, promoting metastatic potential

Anti-proliferative effects:

• NT-4 inhibits cell proliferation while promoting cellular apoptosis in vitro

• It inhibits xenograft tumorigenicity in vivo

• NT-4 increases ANXA1 phosphorylation and sumoylation and its interaction with importin β

• This leads to nuclear import and retention of ANXA1, which activates the caspase-3 apoptosis cascade

Clinical significance:

This dual role suggests that NT-4 may contribute to early metastasis of breast cancer and could serve as a prognostic marker.

How are NTF4 mutations associated with primary open-angle glaucoma?

Studies have identified multiple heterozygous mutations in the NTF4 gene associated with primary open-angle glaucoma (POAG):

Mutation profile:

• Seven different heterozygous mutations in the NTF4 gene (C7Y, E84K, A88V, R90H, R206W, R206Q, and R209G) have been identified in POAG patients

• These mutations account for approximately 1.7% of POAG cases

• The mutations were found in both late juvenile and adult-onset POAG, with age at diagnosis varying from 36 to 80 years

• Most patients with NTF4 mutations had elevated intraocular pressure (IOP) ranging from 25 to 40 mmHg, though some had normal pressure readings

Functional impact:

• Structural analysis and molecular modeling showed that these mutations affect the NT-4 protein structure and its interaction with TrkB

• The mutations were found to impair NT-4 signaling, which may affect retinal ganglion cell survival

• This supports the hypothesis that factors compromising neuronal survival, rather than just elevated intraocular pressure, contribute to POAG pathogenesis

Evidence strength:

These findings provide strong genetic evidence linking NTF4 variants to POAG and highlight the importance of neurotrophic factors in glaucoma pathogenesis.

How does NT-4 function in neuronal development and survival compared to other neurotrophins?

NT-4 plays distinct but overlapping roles with other neurotrophins in neuronal development and survival:

Developmental roles:

• Studies comparing TrkB−/− and Bdnf−/−/Ntf4−/− mice show that NT-4 contributes to geniculate ganglion development

• At E11.5, neuron numbers were reduced by 31% in Bdnf−/−/Ntf4−/− mice compared to wild-type

• By E12.5, neuron counts were reduced by 48% in Bdnf−/−/Ntf4−/− mice and 81% in TrkB−/− mice

• By E13.5, neuron numbers were reduced by 80% in Bdnf−/−/Ntf4−/− mice and 87% in TrkB−/− mice

• These findings suggest that NT-4 and BDNF together account for most, but not all, TrkB-dependent neuronal development

Functional specificity:

• Despite activating the same TrkB receptor as BDNF, NT-4 has unique functions

• NT-4 promotes dendritic outgrowth and calcium currents in cultured mesencephalic dopamine neurons

• It promotes growth and remodeling of adult motor neuron innervation

• NT-4 serves as an anterograde survival factor for postsynaptic cells and protects against apoptotic neuronal death

Signaling mechanisms:

• NT-4 binds to TrkB with high affinity, inducing receptor dimerization and activation

• It also binds to p75NTR, which can modulate TrkB signaling

• NT-4-induced TrkB signaling can augment NMDA receptor activity, potentially increasing neuronal sensitivity to excitotoxic cell death

• These mechanisms contribute to both pro-survival and pro-apoptotic effects depending on the cellular context

What experimental models are most appropriate for studying NT-4 functions in different contexts?

Different experimental models offer advantages for specific aspects of NT-4 research:

In vitro neuronal models:

• Primary neuron cultures (especially mesencephalic dopamine neurons) for studying NT-4's effects on neuronal survival, neurite outgrowth, and electrophysiological properties

• TrkB-transfected cell lines (e.g., HEK-293) for studying receptor activation and signaling pathways

• Organotypic brain slice cultures for studying NT-4's effects in a more intact neural circuit environment

Cancer cell models:

• Breast cancer cell lines for studying NT-4's dual role in proliferation/apoptosis and metastasis

• Xenograft models in immunodeficient mice to evaluate NT-4's effects on tumor growth and metastasis in vivo

• Patient-derived organoids to assess NT-4's effects in a more clinically relevant context

Genetic models:

• TrkB−/− and Bdnf−/−/Ntf4−/− knockout mice for studying developmental roles

• Conditional knockout or knockin models to study tissue-specific or temporal NT-4 functions

• CRISPR/Cas9-engineered cell lines with specific NT-4 pathway mutations

Glaucoma models:

• Transgenic mice expressing human NTF4 mutations identified in POAG patients

• Ex vivo retinal explant cultures for studying NT-4's effects on retinal ganglion cell survival

• Induced pluripotent stem cell (iPSC)-derived retinal organoids from POAG patients with NTF4 mutations

The choice of model should align with the specific research question and consider the expression of relevant receptors and signaling molecules.

How should I design experiments to distinguish between NT-4 and BDNF functions given they share the same TrkB receptor?

Distinguishing between NT-4 and BDNF functions requires careful experimental design:

Receptor-based approaches:

• Utilize specific blocking antibodies that selectively inhibit NT-4 or BDNF binding to TrkB

• Design peptide competitors that mimic specific binding epitopes unique to each neurotrophin

• Compare signaling kinetics and duration by time-course analysis after stimulation with equivalent concentrations of NT-4 versus BDNF

Genetic approaches:

• Use NT-4-specific knockout models compared to BDNF-specific knockouts

• Employ RNA interference targeting either NT-4 or BDNF specifically

• Analyze phenotypes in Bdnf−/−/Ntf4−/− double knockout versus single knockouts to identify unique versus overlapping functions

Functional assays:

• Compare concentration-response relationships for both neurotrophins across multiple endpoints

• Examine context-dependent effects in different cell types that may respond differently to each neurotrophin

• Analyze differential effects on specific TrkB phosphorylation sites or downstream signaling pathways

Expression analysis:

• Compare temporal and spatial expression patterns of NT-4 and BDNF in your experimental system

• Use in situ hybridization to identify cells expressing one or both factors

• Examine co-expression with TrkB to identify potential autocrine/paracrine signaling mechanisms

What are the critical considerations when investigating NT-4's role in disease pathogenesis?

When investigating NT-4's role in disease:

Control for genetic background effects:

• When using transgenic or knockout models, ensure appropriate genetic background controls

• Consider using multiple founder lines or different genetic backgrounds to confirm phenotypes

• For human studies, account for population stratification in genetic association analyses

Consider developmental timing:

• NT-4 has important developmental roles, so distinguishing between developmental versus acute effects is critical

• Use inducible systems (e.g., tamoxifen-inducible Cre-loxP) to control the timing of NT-4/TrkB manipulation

• Include detailed temporal analyses to determine when NT-4 signaling becomes disrupted in disease models

Account for compensatory mechanisms:

• Loss of NT-4 may trigger compensatory upregulation of other neurotrophins

• Assess levels of related neurotrophins (especially BDNF) and their receptors

• Consider using acute versus chronic manipulations to distinguish primary from compensatory effects

Address cell type specificity:

• NT-4 affects multiple cell types beyond neurons, including immune cells and epithelial cells

• Use cell type-specific promoters for targeted manipulation of NT-4 signaling

• Employ methods like single-cell RNA-seq to identify cell populations differentially affected by NT-4

Validate in human samples:

• Confirm findings from model systems in relevant human tissue samples

• Use patient-derived cells or organoids when possible

• Consider NT-4 polymorphisms and mutations identified in human diseases as experimental variables

What are common pitfalls when working with recombinant NT-4 and how can they be avoided?

Common technical challenges with recombinant NT-4 include:

Loss of activity during storage/handling:

• Avoid repeated freeze-thaw cycles by storing as single-use aliquots

• Add carrier protein (0.1 mg/ml BSA) for diluted solutions to prevent adsorption to tubes

• Always centrifuge before use (10,000 × g for 5 min) to remove aggregates

• Validate activity of each lot before use in critical experiments

Batch-to-batch variability:

• Include positive controls with known activity in each experiment

• Consider testing multiple lots or sources when initiating new studies

• Establish internal standards and functional validation protocols

• Quantify activity rather than relying solely on protein concentration

Receptor cross-reactivity:

• NT-4 activates both TrkB and p75NTR receptors

• Include appropriate receptor blocking controls to determine specific receptor contributions

• Consider using receptor-selective mutants or inhibitors when studying specific signaling pathways

• Verify receptor expression in your experimental system before interpreting NT-4 effects

Species differences:

• Human NT-4 shares 91% and 95% amino acid identity with mouse and rat NT-4, respectively

• While highly conserved, these differences may affect cross-species activities

• When possible, use species-matched NT-4 for your experimental system

• Be cautious when interpreting evolutionary studies or cross-species experiments

How can I optimize NT-4 concentration and treatment duration for different experimental endpoints?

Optimization strategies for NT-4 treatments:

Concentration optimization:

• Perform concentration-response experiments covering a wide range (typically 0.1-100 ng/mL)

• The effective concentration for neuronal effects is typically 0.3-3 ng/mL, but this varies by assay

• Include both low (sub-threshold) and high (potentially saturating) concentrations

• Consider receptor density in your system - higher expression may require different concentrations

Treatment duration:

• For signaling studies (e.g., TrkB phosphorylation), use short timepoints (5-60 minutes)

• For neurite outgrowth, longer treatments (24-72 hours) are typically needed

• For survival effects, both acute (24-48 hours) and chronic (5-14 days) treatments may be informative

• Consider pulse treatments versus continuous exposure to model physiological conditions

Experimental design considerations:

• Include time-matched controls for each concentration tested

• For long-term treatments, consider media replenishment schedules and protein stability

• When comparing NT-4 with other neurotrophins, match molar concentrations rather than weight/volume

• For complex endpoints (e.g., gene expression), perform detailed time-course experiments

Validation approach:

• Establish clear quantitative readouts for activity (e.g., % survival, neurite length)

• Use multiple complementary assays to confirm effects (e.g., morphological plus biochemical)

• Include positive controls (e.g., BDNF) and negative controls in each experiment

• Document lot-specific activity for reproducibility across experiments

What are promising new applications of NT-4 in regenerative medicine and neurological disorders?

Emerging research directions for NT-4 include:

Neuroprotective strategies:

• NT-4's ability to promote neuronal survival makes it a candidate for treating neurodegenerative disorders

• Studies suggest potential applications in protecting retinal ganglion cells in glaucoma, especially given the association of NTF4 mutations with POAG

• The distinct properties of NT-4 versus BDNF may provide advantages in specific neuroprotective contexts

Neural tissue engineering:

• NT-4 can be used to enhance the neurotrophic capacity of stem cells for neural tissue engineering

• Combined with other growth factors, NT-4 contributes to axonal growth in engineered neural tissues

• Integration of NT-4 into biomaterials may provide sustained delivery for nerve regeneration

Cancer therapeutics:

• The dual role of NT-4 in cancer (promoting metastasis while inhibiting proliferation) presents complex but potentially valuable therapeutic opportunities

• Targeting NT-4-mediated signaling pathways might be effective in preventing early metastasis

• The prognostic value of NT-4 expression suggests potential applications in personalized medicine approaches

Immune modulation:

• NT-4 is secreted by activated T cells and granulocytes at sites of inflammation

• Its role in tissue regeneration in inflammatory contexts suggests potential applications in immune-mediated diseases

• Further research into NT-4's immune functions may reveal novel therapeutic applications

How does heterodimer formation between NT-4 and other neurotrophins affect biological activity?

NT-4 can form heterodimers with other neurotrophins, which has important functional implications:

Heterodimer formation:

• NT-4 can form heterodimers with BDNF or NT-3, in addition to forming homodimers

• The mature protein is secreted as a homodimer but can also associate with other neurotrophins under physiological conditions

Functional consequences:

• Heterodimers may exhibit unique receptor binding properties compared to homodimers

• They could potentially activate different combinations of Trk receptors simultaneously

• This might allow for more complex signaling outcomes than possible with homodimers alone

Research considerations:

• When studying NT-4 in systems expressing multiple neurotrophins, consider possible heterodimer effects

• Experimental designs should account for endogenous neurotrophin expression

• Purified heterodimers versus homodimers can be used to dissect specific signaling outcomes

Technical approaches:

• Co-expression systems can be used to generate neurotrophin heterodimers

• Biochemical techniques such as co-immunoprecipitation can detect heterodimer formation

• Binding studies with purified heterodimers can assess receptor interactions and activation potential

This area represents an emerging frontier in neurotrophin research with potential implications for both basic science and therapeutic applications.