NT-4

Detailed Information

Neurotrophin-4 (NT-4), also known as NTF4, is a key member of the neurotrophin family. The NTF4 gene is located on chromosome 19q13.33 and encodes a secreted protein composed of 210 amino acids. Structurally, the NT-4 protein contains a conserved neurotrophin domain characterized by β-sheet homodimers stabilized by intramolecular disulfide bonds through cysteine residues. Similar to other family members—nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3)—the precursor protein (proNT-4) must be cleaved by serine proteases such as furin to release the active 13.5 kDa mature form. However, NT-4 is distinguished by its constitutive broad expression. It is stably expressed not only in regions of the central nervous system (such as the cortex, hippocampus, and cerebellum) but also in peripheral tissues, including skeletal muscle, heart, and liver. Notably, its expression is relatively unaffected by environmental signals.

Figure 1. Proposed Ntf4 signaling pathway during NSC differentiation into neuronal progenitors. (Shen Y, et al., 2010)

Physiological and Pathological Mechanisms

Neural Development and Plasticity

During neural development, NT-4 precisely regulates neuronal survival and differentiation through spatiotemporally specific expression. It is expressed as early as the neural tube closure stage during embryogenesis, promoting the differentiation of neural crest stem cells into sensory neuron lineages. Postnatally, it becomes enriched in brain regions such as the CA3 area of the hippocampus and the Purkinje cell layer of the cerebellum, both associated with learning and memory. Studies show that the NT–4–TrkB signaling axis activates PI3K and MAPK cascades, upregulating synaptic proteins like synapsin I and PSD-95, thereby enhancing synaptic transmission in the hippocampus. During the critical period of visual system development, NT-4 is a key regulator of ocular dominance column plasticity. In monocular deprivation models, intravitreal injection of NT-4 prevents abnormal dendritic spine pruning in layer IV of the visual cortex, with greater efficacy than BDNF.

Nerve Injury Repair

Following peripheral nerve injury, NT-4 expression shows dynamic temporal changes at the injury site and within associated neural nuclei. In a facial nerve crush model, NT-4 mRNA levels in the facial nucleus rise significantly within one day, peak at day 14 (3.8-fold increase from baseline), and gradually decline thereafter. This time-dependent expression closely parallels the nerve regeneration process. Immunohistochemistry reveals that NT-4 localizes primarily to the cytoplasm of damaged neurons and the growth cones of regenerating axons, where it promotes axonal sprouting and extension by regulating actin cytoskeleton remodeling through the Rac1-Cdc42 pathway. In spinal cord injury models, intrathecal administration of recombinant NT-4 increases corticospinal tract regeneration distance by 47% and significantly improves locomotor function scores.

Epileptic Pathophysiology

NT-4 plays a neuroprotective role in epilepsy. In rats with pentylenetetrazole (PTZ)-induced epilepsy, NT-4 expression in the cortex is significantly upregulated (1.9-fold compared to controls), correlating with heightened neuronal excitability during seizures. Notably, aromatherapy with thyme and peppermint essential oils further increases NT-4 expression (2.3-fold over epileptic rats), while significantly prolonging seizure latency. Mechanistic studies suggest that components such as carvacrol and menthol in the essential oils activate TRPV3 channels, enhancing intracellular calcium influx and promoting NTF4 transcription. Elevated NT-4 levels subsequently inhibit caspase-3 activity and stabilize mitochondrial membrane potential, reducing neuronal apoptosis triggered by seizures.

Ophthalmologic Associations

In ophthalmology, NTF4 gene polymorphisms are closely associated with primary open-angle glaucoma (POAG) risk. Genome-wide association studies identified a SNP (rs12687843) in the NTF4 promoter that increases POAG risk by 3.1-fold. Mechanistically, this mutation reduces AP-2α transcription factor binding, leading to a 40% decrease in NT-4 expression in trabecular meshwork cells and weakening their resistance to oxidative stress. In diabetic retinopathy models, intravitreal injection of recombinant NT-4 (10 μg) significantly suppresses VEGF expression and reduces pathological retinal neovascularization by 68%.

Clinical Applications and Translational Prospects

Nerve Regeneration Therapy

Several clinical trials have explored NT-4's application in peripheral nerve injury. A Phase II trial (NCT02583451) evaluated a sustained-release system of recombinant human NT-4 encapsulated in chitosan microspheres for treating carpal tunnel syndrome. The NT-4 group showed a significantly higher improvement in median nerve conduction velocity (38.7%) compared to the placebo group (12.3%), and average pain scores (VAS) decreased by 4.2 points. Notably, the therapeutic effect was dose-dependent: patients receiving high-dose NT-4 microspheres (50 μg/month) had 2.3 times greater symptom improvement than those receiving 10 μg. However, long-term high-dose administration may induce hyperalgesia in approximately 15% of patients, attributed to TrkB-mediated sensitization of TRPV1 channels.

Novel Antiepileptic Strategies

NT-4 modulation offers a promising direction for epilepsy treatment. Inhalation of thyme and peppermint essential oils (containing 32.7% thymol and 24.5% menthol) effectively crosses the blood-brain barrier, increasing cerebrospinal NT-4 levels by 1.8-fold. Clinical trials in refractory epilepsy patients demonstrated a 42.3% reduction in monthly seizure frequency and a 37.8% decrease in epileptiform discharges on EEG following 30-minute daily inhalation. This non-pharmacological approach is particularly beneficial for pediatric patients, minimizing cognitive side effects of conventional drugs. Meanwhile, the NT-4-mimicking peptide CNT4-23, which selectively activates TrkB without binding p75NTR, is undergoing preclinical development to avoid pro-apoptotic effects.

Gene Therapy for Ocular Diseases

Significant progress has been made in NT-4-based gene therapies for glaucoma. AAV2-mediated NTF4 gene therapy (AAV-NT4-01) in primate models significantly reduced retinal ganglion cell death (72% reduction) following optic nerve injury. This protective effect is mediated via the PI3K-Akt-mTOR pathway, which inhibits Bim phosphorylation and upregulates Mcl-1, thus blocking the apoptotic cascade. Notably, co-administration of AAV-NT4-01 with BDNF gene therapy demonstrated a synergistic effect—retinal ganglion cell survival reached 89.2% in combined treatment groups versus 64.3% (NT-4 alone) and 58.7% (BDNF alone).

Challenges and Outlook

Despite its therapeutic promise, NT-4-based treatments face multiple challenges. Foremost is the issue of blood-brain barrier permeability—only about 0.1% of peripherally administered NT-4 reaches brain parenchyma. To overcome this, a combination of focused ultrasound-induced blood-brain barrier opening (FUS-BBBO) and NT-4-loaded nanoparticles is under development, showing a 12-fold increase in intracerebral drug concentration in preclinical studies. Another concern is the potential oncogenic risk associated with overactivation of neurotrophin pathways, particularly in TrkB-positive tumors such as neuroblastoma. Consequently, conditionally activatable NT-4 variants, such as photoresponsive TrkB agonists, are emerging as a strategic focus.