BLF stimulates neuronal differentiation via activation of p35/CDK5 signaling and AMPK-mediated mitochondrial regulation

Abstract

Lactoferrin (LF) is a multifunctional glycoprotein with established roles in non-neuronal cell growth and differentiation and has underexplored potential in neurodevelopment. Here, we investigated bovine lactoferrin (bLF) as a neurotrophic agent, systematically evaluating its effects on neuronal differentiation, morphology, and mitochondrial regulation in PC12 cells. We demonstrated that bLF (50 μg/mL) induces neurite outgrowth comparable to nerve growth factor (NGF) while maintaining >90 % cell viability. Mechanistically, bLF activated TrkA by phosphorylation at Ser490, followed by ERK phosphorylation at Thr202/Tyr204 within 60 min, mirroring canonical NGF signaling. bLF also upregulates p35 (CDK5 activator) and phosphorylates Synapsin-I, driving presynaptic maturation. Structurally predicted to bind TrkA's ligand-binding interface, bLF synergizes with NGF to amplify differentiation outcomes. Furthermore, TMRE staining and AMPK phosphorylation assays revealed that bLF enhances axonal mitochondrial activity, surpassing NGF's effects. These results establish bLF as a multifunctional neurotrophic agent that coordinates TrkA-ERK-p35/CDK5 signaling, synaptic protein activation, and AMPK-driven mitochondrial regulation. Given its safety profile and synergy with endogenous neurotrophic pathways, bLF emerges as a promising candidate for neuroregenerative therapies targeting nerve injury or neurodegeneration.

Keywords: AMPK activation; Bovine lactoferrin (BLF); Mitochondrial function; Neuronal differentiation; TrkA-ERK signaling; p35/CDK5 activation.