Gambogic amide, a selective agonist for TrkA receptor that possesses robust neurotrophic activity, prevents neuronal cell death

Abstract

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.

Fig. 1.

The juxtamembrane domain of TrkA binds GA. (A) The extracellular domain-truncated TrkA mutants potently bind to gambogic amide. The expression of transfected various GFP-TrkA mutants by immunoblotting with anti-GFP antibody. (B) Some of the ICD-truncated TrkA mutants, including the whole ICD (ΔICD) and SHC-binding domain- deleted (ΔSHC) mutants, failed to bind GA. (C) Sequence alignment of the ICD of TrkA, TrkB, and TrkC. (D) Gambogic amide selectively bound to TrkA, but not TrkB or TrkC receptor. (Upper) The tyrosine kinase-dead TrkA decreased its binding affinity to gambogic amide. (Lower) The expression of transfected constructs was verified. (E) Competition assay for the K_d. GFP-TrkA-associated gambogic amide beads were incubated with increased gambogic amide and various GA derivatives. After extensive washing, the beads-bound proteins were monitored by immunoblotting and quantitated by National Institutes of Health Image software. The K_d for the gambogic amide to TrkA is ≈75 nM. The inactive control GA derivatives did not compete for binding at all (Student's t test; *, P < 0.05). (F) FITC-gambogic amide penetrates cell membrane and binds to TrkA receptor. PC12 cells were incubated for 10 min with 0.5 μM FLTC-gambogic amide and control FITC, respectively. (Upper) After washing and fixing, the cells were stained with TrkA antibody. (Lower) Depletion of SHC drug-binding domain abolishes GA-FITC membrane penetration.

Fig. 2.

Gambogic amide protects hippocampal neurons from apoptosis. (A) (Left) Chemical structures of GA derivatives. (Right) The chemical structure of gambogic amide with the numeric positions labeled. (B) (Left) Some GA derivatives prevent apoptosis in TrkA-expressing T17 cells, but not control SN56 cells. (Right) Interestingly, dimethyl-GA and acetyl iso-GA also protect TrkB-stable cells from apoptosis as BDNF. (C) Gambogic amide exhibits the strongest antiapoptotitc activity in T17 cells. EC₅₀ values for GA derivatives in preventing apoptosis in T17 cells are 10 nM for gambogic amide, 50 nM for dihydro-GA, 55 nM for GA, and 750 nM for dimethyl-GA. (D) Gambogic amide protects hippocampal neurons from glutamate-triggered apoptosis. (E) (Left) Gambogic amide protects neurons from apoptosis triggered by OGD. (Right) Gambogic amide displayed a dose-dependent protective manner on neurons in OGD. All results from three independent experiments were expressed as mean ± SD (Student's t test; *, P < 0.05).

Fig. 3.

Gambogic amide elicits TrkA tyrosine phosphorylation in hippocampal neurons. (A) Gambogic amide induces TrkA tyrosine phosphorylation in primary neurons. Immunoblotting analysis with anti-phospho-Trk490 of hippocampal neurons treated with 0.5 μM GA derivatives for 30 min. (B) Immunofluorescent staining of GA derivative-treated hippocampal neurons with anti-phospho-Trk490 antibody. Both NGF and gambogic amide selectively triggered TrkA Y490 phosphorylation in neurons. (C) K252a decreases gambogic amide-triggered TrkA tyrosine phosphorylation. (D) Gambogic amide provokes TrkA dimerization. GFP-TrkA and HA-TrkA or HA-TrkB were, respectively, cotransfected into HEK293 cells and treated with 0.5 μM GA or gambogic amide for 30 min. GFP-TrkA was immunoprecipitated with anti-GFP antibody, and the coprecipitated proteins were analyzed with anti-HA antibody. Gambogic amide, but not GA, evidently stimulated TrkA dimerization. The stimulatory effect was even stronger than NGF. (Upper) By contrast, TrkA did not bind to TrkB regardless of NGF or gambogic amide treatment. (E) Gambogic amide triggered TrkA, but not TrkB or TrkC, tyrosine phosphorylation in transfected HEK293 cells. Kinase-dead TrkA displayed negligible tyrosine phosphorylation. (F) In vitro binding assay with GST-Trk recombinant proteins. Gambogic amide, but not other GA derivatives, selectively binds TrkA, but not TrkB or TrkC. (G) GA derivatives are unable to trigger both PI3-kinase and MAPK signaling activation in TrkB-stable cells. The experiments and immunoblotting gels in this study were repeated more than three times.

Fig. 4.

Gambogic amide provokes MAPK and Akt kinase activation and neurite outgrowth. (A) Gambogic amide selectively provokes Erk phosphorylation in T17 cells, whereas other derivatives exhibited weakly stimulatory effects. By contrast, in addition to gambogic amide, GA and decahydro-GA also triggered robust Akt activation. (B) GA derivatives initiate Akt activation in hippocampal neurons. (C) Characterization of gambogic amide's stimulatory effect on Akt phosphorylation. (Left) Then 500 nM gambogic amide elicited Akt activation in a time-dependent manner. (Right) Gambogic amide induced Akt activation in a dose-dependent manner during 30-min treatment. (D) PC12 cells were treated with NGF or 0.5 μM GA derivatives for 5 days. Gambogic amide triggered neurite outgrowth as potently as NGF, and dihydro-GA also displayed weak stimulatory activity. By contrast, other derivatives had no effect. (E) Dose-dependent effect of neurite outgrowth. Further, 10–50 nM gambogic amide can provoke neurite outgrowth in PC12 cells. (F) K252a abolishes gambogic amide-provoked neurite outgrowth in PC12 cells.

Fig. 5.

Gambogic amide prevents neuronal cell death and decreases infarct volume in MCAO animal model. (A) Gambogic amide diminished Kainic acid-triggered hippocampal neuronal cell death. The brain slides were analyzed with TUNEL assay and stained with DAPI. Green stands for apoptotic nuclei, which also were stained with DAPI. Kainic acid evidently initiated devastating apoptosis in hippocampal CA3 region, which was substantially blocked by gambogic amide (Upper). (Lower Left) Quantitative analysis of apoptosis in the hippocampal neurons. (Lower Right) KA treatment enhanced TrkA expression in hippocampus and cortex. (B) Gambogic amide reduces infarct volume in MCAO rat brain. Tetanus toxin-stained coronal section from representative animals given either vehicle or gambogic amide and had brains harvested at 24 h postocclusion. (Left) The infarct area in gambogic amide-treated animals is substantially reduced. Infarct volumes after 24 h MCAO. Compared with vehicle alone, gambogic amide significantly reduced total infarct volumes (percentage of contralateral hemisphere). (Right) The data are represented as mean ± SD; *, P < 0.05.