Introduction: Spinal cord injury often results in permanent deficits below the level of the injury. Adult mammalian central nervous system (CNS) has limited inherent capacity to regenerate and restore functional connectivity across the injury site. During development, neurotrophic factors such as Insulin-like growth factor-1 (IGF-1) enhance CNS axon outgrowth. Corticospinal motor neurons express high levels of IGF-1 receptors (IGF-1R) during development. The effect of IGF-1 is mediated via the IGF-IR and its downstream PI3-kinase/Akt pathway. In mature CNS, IGF-1R was downregulated on the CNS axons and results in limited regeneration from the mature axons. Through a screening experiment, we identified osteopontin (OPN), a secreted, glycosylated phosphoprotein, enhances the IGF-1 function and promotes mature neuron axon regeneration combined with IGF-1.
Methods: We used cell culture and optic nerve regeneration system to study the molecular mechanism of the synergistic effect of OPN with IGF-1 for axon regeneration.
Results: Using the primary cortical neuron cell culture, OPN plus IGF-1, but neither OPN or IGF-1 alone, promotes IGF-1R phosphorylation (activated form of IGF-1R) and the activation of its downstream PI3K pathway. Phosphorylation of the ß-subunit of IGF1R is a critical step to inducing neurite outgrowth by IGF1. OPN or its N-terminal segment, but not the C-terminal segment, promotes significant neurite outgrowth in primary neuron culture.
We identified that OPN faciliates IGF-1R activation by enabling it to be enriched into distinct cholesterol-rich membrane microdomain (lipid raft). This process is likely mediated with integrin, as the deletion of the OPN integrin binding site abolish the effect of OPN in promoting IGF-1/IGF-1R signaling. Using co-immunoprecipitating experiment, we showed that OPN promote the binding of IGF-1R to the ß1 and ß3 integrins. Similar OPN enhancing effect is observed in other neurotrophin pathway such as BDNF/TrkB pathway. Using optic nerve injury model, OPN with IGF-1 but neither OPN or IGF-1 alone induces significant optic nerve regeneration after complete crush injury.
Conclusions: OPN promotes neurotrophin pathways such as IGF-1/IGF-1R signaling in adult CNS axon regeneration. It's effect is through the enrichment of neurotrophin receptors and integrins into specific membrane micro-domains, which leads to the neurotrophin pathway activation within the injured axons.
Patient Care: This research provides molecular mechanism and potential therapeutic strategies for repair after CNS injury such as spinal cord injury.
Learning Objectives: By the conclusion of this session, participants should be able to: 1) recognize the role of osteopontin in promoting the neurotrophin axon regeneration effect. 2) understand the mechanism is through the effect of enrichment of neurotrophin receptors and integrins onto specific membrane microdomain. 3) understand the potential role of osteopontin on the spinal cord injury repair.