Nerve growth factor (NGF) binds to TrkA receptor and triggers activation of numerous signaling cascades, which play critical roles in neuronal plasticity, survival, and neurite outgrowth. To mimic NGF functions pharmacologically, we developed a high-throughput screening assay to identify small-molecule agonists for TrkA receptor. The most potent compound, gambogic amide, selectively binds to TrkA, but not TrkB or TrkC, and robustly induces its tyrosine phosphorylation and downstream signaling activation, including Akt and MAPKs. Further, it strongly prevents glutamate-induced neuronal cell death and provokes prominent neurite outgrowth in PC12 cells. Gambogic amide specifically interacts with the cytoplasmic juxtamembrane domain of TrkA receptor and triggers its dimerization. Administration of this molecule in mice substantially diminishes kainic acid-triggered neuronal cell death and decreases infarct volume in the transient middle cerebral artery occlusion model of stroke. Thus, gambogic amide might not only establish a powerful platform for dissection of the physiological roles of NGF and TrkA receptor but also provide effective treatments for neurodegenerative diseases and stroke.
Toll-like receptor 4 (TLR4) contributes to cerebral ischemia/reperfusion (I/R) injury and is a potential target for the treatment of ischemic stroke. This experiment is to evaluate the effect of an exogenous TLR4 antagonist, TAK-242, against acute cerebral I/R injury. A mouse model of cerebral I/R was induced by transient middle cerebral artery occlusion. TAK-242 (3 mg/kg body weight) was injected intraperitoneally 1 hour after ischemia. Our results showed that the concentration of TAK-242 in plasma increased to 52.0 ng/mL 3 hours after injection, was maintained at 54.1 ng/mL 8 hours after injection, and decreased to 22.6 ng/mL 24 hours after injection. The concentration of TAK-242 in brain tissue increased to 26.1 ng/mL in ischemic hemisphere and 14.2 ng/mL in nonischemic hemisphere 3 hours after injection, and was maintained at the similar levels 24 hours after injection. We found that TAK-242 significantly reduced cerebral infarction compared with vehicle control, improved neurologic function, inhibited the phosphorylation of downstream protein kinases in TLR4 signaling pathway, and downregulated the expression of inflammatory cytokines. We conclude that TAK-242 is able to cross blood-brain barrier, blocks TLR4 signaling, mediates the expression of inflammatory cytokines, and protects the brain from acute damage induced by I/R.