NT 3 Human
Description
Molecular Structure and Biosynthesis
NT-3 is synthesized as a 257-amino acid precursor protein, which undergoes proteolytic cleavage to yield the mature 119-amino acid protein . Key structural features include:
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Homodimer Formation: Bioactive NT-3 exists as a non-covalently linked homodimer (27.3 kDa total mass) .
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Cysteine Knot Motif: Contains six conserved cysteine residues forming three disulfide bonds, creating a twisted four-stranded β-sheet structure .
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Species Conservation: Mature NT-3 sequences are identical in humans, mice, rats, and pigs .
| Feature | Details |
|---|---|
| Precursor Length | 257 amino acids |
| Mature Protein Size | 119 amino acids (13.6 kDa monomer) |
| Structural Motif | Cysteine knot with β-sheet core |
| Dimerization | Non-covalent homodimer |
Receptor Interactions and Signaling Pathways
NT-3 exerts its effects through high-affinity and low-affinity receptors:
Key Signaling Pathways:
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TrkC Activation: NT-3 binding induces receptor dimerization, activating downstream kinases (e.g., MAPK, PI3K/Akt) to regulate neuronal survival .
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p75 Signaling: Binds NT-3 to form a 2:2 ligand-receptor complex, modulating apoptosis or survival depending on cellular context .
Central Nervous System (CNS)
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Hippocampal Neurogenesis:
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Cortical Development: Essential for oligodendrocyte differentiation and cortical organoid maturation .
Peripheral Nervous System (PNS)
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Sensory Neuron Survival: NT-3 deficiency leads to impaired proprioceptive and mechanoreceptive neuron development .
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Cardiovascular Regulation: Supports endothelial and myocardial cell survival .
In Vitro and In Vivo Models
Therapeutic Targets
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Neurodegenerative Diseases: NT-3’s role in neuroprotection and synaptic repair positions it as a candidate for Alzheimer’s and Parkinson’s therapies .
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Peripheral Nerve Regeneration: NT-3’s ability to support sensory neuron survival makes it valuable for treatments of diabetic neuropathy .
Comparative Analysis with Other Neurotrophins
Challenges and Future Directions
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Dose-Dependent Effects: Excessive NT-3 may inhibit proliferation in neurogenic niches (e.g., hippocampal subgranular zone) .
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Delivery Challenges: Systemic administration faces barriers due to blood-brain barrier limitations .
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Synergistic Therapies: Combined use with other neurotrophins (e.g., BDNF) or growth factors (e.g., PDGF-AA) may enhance efficacy .
Product Specs
Q&A
What is Neurotrophin-3 and what distinguishes it from other neurotrophins?
Neurotrophin-3 (NT-3) is a protein growth factor in the NGF (Nerve Growth Factor) family of neurotrophins. It was the third neurotrophic factor to be characterized after NGF and BDNF (Brain Derived Neurotrophic Factor). In humans, NT-3 is encoded by the NTF3 gene . It supports the survival and differentiation of existing neurons while encouraging the growth and differentiation of new neurons and synapses .
What distinguishes NT-3 from other neurotrophins is its unique ability to activate multiple neurotrophin receptors. While most neurotrophins activate primarily one tyrosine kinase receptor, NT-3 can stimulate neurons through both TrkC (its high-affinity receptor) and TrkB receptors, giving it broader neuronal targeting capabilities .
How is the structure of human NT-3 characterized and what significance does the cysteine knot have?
Human NT-3 is a 119-amino acid residue mature protein derived from a 257-amino acid precursor protein that undergoes proteolytic processing . The structural hallmark of NT-3, like all neurotrophins, is the characteristic arrangement of disulfide bridges known as the cysteine knot . This structural motif, also found in other growth factors such as PDGF, is critical for the protein's stability and function .
The cysteine knot configuration enables NT-3 to form a bioactive non-covalently linked homodimer, which is essential for receptor binding and activation . The amino acid sequence of mature NT-3 demonstrates remarkable evolutionary conservation, being identical in human, mouse, and rat, suggesting the critical biological importance of its precise structure .
What are the primary receptors for NT-3 and how does their binding drive downstream signaling?
NT-3 interacts with three main receptors:
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TrkC (Tyrosine receptor kinase C): The primary "physiologic" receptor that binds NT-3 with highest affinity .
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TrkB: A TrkC-related receptor that can also bind and transmit NT-3 signals, though with lower affinity than TrkC .
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LNGFR (Low-affinity nerve growth factor receptor): Also known as p75NTR, which binds all neurotrophins .
When NT-3 binds to TrkC, it activates receptor tyrosine kinase activity, triggering phosphorylation cascades that activate multiple signaling pathways. This includes activation of ERK1/2 MAPK phosphorylation, which can be experimentally observed in TrkC-transfected PC12 cells when treated with recombinant human NT-3 . The activation of these downstream pathways ultimately mediates NT-3's effects on neuronal survival, differentiation, and neurite outgrowth .
How does NT-3 expression vary across brain regions and developmental stages?
NT-3 expression follows distinct spatial and temporal patterns in the brain. During perinatal development, NT-3 shows particularly high expression levels, with most prominent expression in the hippocampus, neocortex, and cerebellum . As development progresses into adulthood, NT-3 expression generally declines in most brain regions .
How do stress and antidepressant treatments affect NT-3 expression in the hippocampus?
Stress and antidepressant treatments have opposing effects on NT-3 expression in the hippocampus:
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Stress conditions: Chronic unpredictable mild stress increases NT-3 expression in the hippocampus . Similarly, corticosterone administration (mimicking stress hormone elevation) increases NT-3 expression specifically in the dentate gyrus .
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Antidepressant treatments: Chronic administration of antidepressants such as fluoxetine or electroconvulsive seizures decreases NT-3 expression in the dentate gyrus of adult mice .
These opposing effects suggest that NT-3 expression may serve as a regulatory mechanism in stress responses, potentially contributing to stress-induced changes in hippocampal function and neurogenesis. The bidirectional regulation implies NT-3 may be part of a homeostatic system responding to environmental challenges and therapeutic interventions.
How does NT-3 regulate different phases of adult neurogenesis in the dentate gyrus?
NT-3 exerts differential effects on distinct phases of adult neurogenesis in the dentate gyrus, with both stimulatory and inhibitory roles depending on expression levels and neurogenic phase:
Differentiation phase: NT-3 overexpression reduces the number of doublecortin (DCX)-positive immature neurons in the SGZ, affecting both cells with short dendrites and those with long, branched dendrites . This suggests NT-3 regulates the early differentiation and morphological development of newborn neurons.
Survival phase: Interestingly, NT-3 overexpression does not significantly affect the survival rate of BrdU-labeled cells assessed 31-33 days after labeling . This contrasts with findings from NT-3 deficiency models, where neuronal differentiation and survival (rather than proliferation) are impaired, suggesting complex dose-dependent effects.
What molecular mechanisms mediate NT-3's effects on neurogenesis?
NT-3's effects on neurogenesis appear to be mediated through several molecular mechanisms:
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TrkC receptor signaling: TrkC is expressed in the granule cell layer (GCL) and hilus regions of the dentate gyrus. NT-3 regulates downstream signaling via TrkC, though prolonged NT-3 overexpression can induce feedback regulation resulting in decreased TrkC expression .
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ERK signaling pathway: NT-3 overexpression tends to increase phosphorylated ERK (pERK) in scattered cells within the granule cell layer, suggesting activation of this classical neurotrophin signaling pathway . ERK activation likely contributes to NT-3's effects on neuronal differentiation and maturation.
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Neurogenesis-related gene expression: NT-3 overexpression has been shown to significantly decrease the expression of several neurogenesis-related factors, including Vegfd, Lgr6, Bmp7, and Drd1 . These transcriptional changes likely contribute to the suppression of early neurogenic processes.
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Regulation of mature neuronal activation: High NT-3 levels in the hippocampus regulate the activation of mature dentate gyrus neurons as evidenced by changes in the expression of immediate early genes such as Fos and Fosb .
What are the optimal methods for studying NT-3 signaling in neuronal cultures?
When studying NT-3 signaling in neuronal cultures, researchers should consider the following methodological approaches:
Recombinant protein selection: Use high-quality recombinant human NT-3 protein. Options include E. coli-derived protein or Spodoptera frugiperda Sf21 (baculovirus)-derived human NT-3 protein (amino acids Tyr139-Thr257) with potential modifications like K196R . The choice depends on the specific application and required purity.
Dose determination: For neurite outgrowth assays, a concentration of 30 ng/ml of recombinant human NT-3 has been validated in TrkC-transfected PC12 cells . For ERK1/2 MAPK phosphorylation studies, a concentration range of 4.00-40.0 ng/ml is typically effective .
Experimental timeframes:
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For neurite outgrowth assessment: Allow 5 days after NT-3 treatment for visible neurite extension
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For signaling pathway activation: Examine ERK1/2 phosphorylation after 8 minutes of NT-3 stimulation following 3 hours of serum deprivation
Cell preparation: Prior to NT-3 treatment, cells should undergo appropriate transfection with TrkC receptors (if using PC12 or similar model systems), followed by serum deprivation for 3 hours to reduce baseline signaling activity .
What are the most effective in vivo approaches for manipulating NT-3 levels in specific brain regions?
Several approaches have proven effective for manipulating NT-3 levels in specific brain regions:
Viral vector-mediated overexpression: Adeno-associated virus (AAV) vectors expressing NT-3 can be stereotaxically injected into specific brain regions. This approach has been successfully used to overexpress NT-3 in the dentate gyrus, resulting in significantly increased NT-3 protein levels . This method allows for region-specific manipulation with sustained expression.
Genetic approaches: Conditional knockout models using Cre-lox technology (e.g., Nestin-Cre line to suppress NT-3 expression specifically in the brain) allow for tissue-specific manipulation of NT-3 levels . This approach is particularly useful for studying the consequences of NT-3 deficiency.
Pharmacological modulation: Administration of agents known to alter NT-3 expression, such as antidepressants (fluoxetine) or corticosterone, can be used to indirectly manipulate NT-3 levels . While less specific than genetic approaches, this method can mimic physiological or pathological conditions.
Neurogenesis assessment: Following NT-3 manipulation, researchers can assess neurogenesis using:
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EdU labeling (2 hours before sacrifice) to quantify proliferating cells
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DCX immunostaining to identify immature neurons
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BrdU pulse-chase experiments with 31-33 day intervals to examine cell survival
How does NT-3 contribute to stress responses and hippocampal function?
NT-3 appears to play a critical role in stress responses and hippocampal function through several mechanisms:
Regional specificity in stress processing: The hippocampus has functionally distinct roles in its dorsal and ventral regions. The dorsal region is involved in stress-induced learning and memory changes, while the ventral region contributes to stress response and emotions like anxiety . NT-3 is expressed in both regions and may differentially regulate these functions.
Neuronal activation regulation: High NT-3 levels in the hippocampus regulate the activation of mature dentate gyrus neurons, as evidenced by changes in immediate early gene expression (Fos and Fosb) . This suggests NT-3 modulates neuronal excitability and activity patterns in response to environmental stimuli.
Bidirectional regulation under stress: NT-3 expression is increased in the hippocampus following chronic unpredictable mild stress and after corticosterone administration . Conversely, antidepressant treatments that mitigate stress effects decrease NT-3 expression . This bidirectional regulation suggests NT-3 may be part of an adaptive response mechanism to stress conditions.
Neurogenesis suppression: Elevated NT-3 levels suppress early phases of adult neurogenesis , which may represent a mechanism for stress-induced cognitive changes, as neurogenesis in the ventral dentate gyrus has been linked to stress resilience .
What is the current understanding of NT-3's potential role in neurological disorders?
While the search results don't directly address NT-3's role in specific neurological disorders, several implications can be drawn from its known functions:
Stress-related disorders: Given NT-3's responsiveness to stress conditions and antidepressant treatments, it may play a role in stress-related disorders such as depression and anxiety. The bidirectional regulation of NT-3 by stress and antidepressants suggests potential involvement in the pathophysiology or treatment mechanisms of these conditions .
Neurodevelopmental implications: NT-3 is essential for the survival of peripheral sensory and sympathetic neurons during development . Mice born without NT-3 show loss of proprioceptive and subsets of mechanoreceptive sensory neurons . This suggests potential implications for sensory processing disorders or developmental neurological conditions.
Neuroplasticity and recovery: NT-3 strengthens synaptic connections to motoneurons in neonatal rats and serves as an anti-inflammatory factor suppressing microglial activation . These properties suggest potential roles in neurorehabilitation, recovery from neural injury, or neurodegenerative disorders where inflammation plays a key role.
Hippocampal dysfunction: Given NT-3's specific roles in hippocampal function and adult neurogenesis, it may be implicated in disorders affecting hippocampal-dependent cognitive functions, including memory disorders and certain aspects of neurodegenerative diseases.
Product Science Overview
NT-3 is a protein that is highly conserved across species, with identical amino acid sequences in humans, mice, and pigs . It exists as a monomer with a molecular weight of approximately 13.6 kDa or as a dimer with a molecular weight of 27.2 kDa . The protein is involved in the development and maintenance of neuronal populations and promotes the differentiation of neural crest-derived sensory and sympathetic neurons .
NT-3 exerts its effects by binding to and activating specific receptors on the surface of target cells. It primarily interacts with the TrkC receptor but can also bind to TrkA and TrkB receptors . Upon binding, these receptors activate several intracellular signaling pathways, including the Ras/MAPK pathway, the PI3 kinase/Akt pathway, and the PLC-γ1/PKC pathway . These pathways are involved in promoting neuronal differentiation, survival, growth, and synaptic plasticity .
NT-3 is expressed in various cell types, including neuronal cells, eosinophils, and melanocytes . It is essential for the development of peripheral sensory neurons and is required for the proper functioning of the nervous system . Knock-out studies in mice have shown that the absence of NT-3 leads to movement and posture deficiencies due to proprioception defects and loss of neurons in spinal and cephalic sensory ganglia .
Recombinant human NT-3 (hNT-3) is produced using recombinant DNA technology, typically in bacterial systems such as E. coli . The recombinant protein is purified to high levels of purity, often greater than 95% . It is used in various research applications to study its effects on neuronal cells and to understand its role in the nervous system.
Recombinant hNT-3 is supplied in a lyophilized form and can be reconstituted with sterile water or buffer solutions . It is stable at -20°C for long-term storage and can be used in various experimental setups to study its biological activity .
