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Amitriptyline is a TrkA and TrkB receptor agonist that promotes TrkA/TrkB heterodimerization and has potent neurotrophic activity - PubMed

  • ️Thu Jan 01 2009

Amitriptyline is a TrkA and TrkB receptor agonist that promotes TrkA/TrkB heterodimerization and has potent neurotrophic activity

Sung-Wuk Jang et al. Chem Biol. 2009.

Abstract

Neurotrophins, the cognate ligands for the Trk receptors, are homodimers and induce Trk dimerization through a symmetric bivalent mechanism. We report here that amitriptyline, an antidepressant drug, directly binds TrkA and TrkB and triggers their dimerization and activation. Amitriptyline, but not any other tricyclic or selective serotonin reuptake inhibitor antidepressants, promotes TrkA autophosphorylation in primary neurons and induces neurite outgrowth in PC12 cells. Amitriptyline binds the extracellular domain of both TrkA and TrkB and promotes TrkA-TrkB receptor heterodimerization. Truncation of amitriptyline binding motif on TrkA abrogates the receptor dimerization by amitriptyline. Administration of amitriptyline to mice activates both receptors and significantly reduces kainic acid-triggered neuronal cell death. Inhibition of TrkA, but not TrkB, abolishes amitriptyline's neuroprotective effect without impairing its antidepressant activity. Thus, amitriptyline acts as a TrkA and TrkB agonist and possesses marked neurotrophic activity.

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Figures

Figure 1
Figure 1. Amitriptyline selectively protects hippocampal neurons from apoptosis

(A) Chemical Structures of tricyclic anti-depressant drugs. (B) Some of the tricyclic anti-depressant drugs protect T17-TrkA cells but not parental SN56 cells from apoptosis. (C) EC50 titration assays for promoting T17 cell survival. TrkA-overexpressing T17 cells were pretreated with various tricyclic antidepressant drugs for 30 min, followed by 1 μM staurosporine for 9 h. Apoptosis was quantitatively analyzed. EC50 values are the drug concentrations, which prevent 50% cells from apoptosis. (D) Amitriptyline selectively prevents apoptosis in hippocampal neurons. Hippocampal neurons were pretreated with NGF (100 ng/ml), gambogic amide and various tri-cyclic antidepressant drugs (0.5 μM) for 30 min, followed by 50 μM glutamate for 16 h. Apoptosis was quantitatively analyzed. (E) Amitriptyline prevents OGD-provoked neuronal apoptosis in hippocampal neurons. Hippocampal neurons were pretreated with various drugs (0.5 μM) for 30 min, followed by OGD for 3 h. Apoptosis was quantitatively analyzed (left panel). Data represent the mean ± SEM of n=4−5; (One-way ANOVA, followed by Dunnett's test, *p<0.01; ** p<0.005).

Figure 2
Figure 2. Amitriptyline activates the TrkA receptor and its downstream signaling cascades

(A) Amitriptyline activates TrkA in hippocampal neurons. Hippocampal neurons were treated with NGF (100 ng/ml) or various tricyclic antidepressant drugs (500 nM) for 30 min. Immunofluorescent staining was conducted with anti-p-TrkA antibody. (B) Amitriptyline activates TrkA downstream signaling cascades including Akt and MAP kinases. Hippocampal neurons were treated as described above. The cell lysates were analyzed by immunoblotting with various antibodies. (C) Trk receptor inhibitor K252a (100 nM) blocks amitriptyline-provoked TrkA and TrkB activation. Data represent mean ± S.E.M (One-way ANOVA, Dunnett's test, *p<0.001). (D) Akt and MAP kinase activation kinetics and dosage assays by amitriptyline. Hippocampal neurons were treated with 500 nM amitriptyline for various time points. The cell lysates were analyzed by immunoblotting with various antibodies.

Figure 3
Figure 3. Amitriptyline provokes neurite outgrowth in PC12 cells

(A) Amitriptyline but not other tricyclic compounds provokes neurite outgrowth in PC12 cells. PC12 cells were treated with amitriptyline and other compounds (500 nM) for 5 days in 2% FBS and 1% HS medium. The drug containing medium was replenished every other day. Amitriptyline induced neurite outgrowth as potently as NGF (upper panel). The relative neurite length was quantified (lower panel). (B) Dose-dependent effect of neurite outgrowth. 100 nM amitriptyline was able to provoke neurite outgrowth in PC12 cells (One-way ANOVA, Bonferroni post hoc test, *p<0.01; **p<0.005). (C) K252a, PI 3-kinase and MAP kinase inhibitors abolish amitriptyline-provoked neurite outgrowth in PC12 cells.

Figure 4
Figure 4. Amitriptyline binds TrkA and TrkB receptors and triggers their dimerization and autophosphorylation

(A) In vitro binding assay with Trk recombinant proteins. 0.1 μCi [3H]-amitriptyline was incubated with purified ECD and ICD recombinant proteins of Trk receptors (5 μg each) at 30 °C for 1 h. The mixture was subjected to filter/vaccum assay. After extensive washing, the filter paper was analyzed in liquid scintillation counter. (B) Quantitative analysis the binding between amitriptyline and TrkA, TrkB ECD. TrkA and TrkB ECD binding curve by [3H]amitriptyline (upper panel). Scatchard plot for Kd constants analysis (lower left). Cold aminitryptyline competed with [3H]amitriptyline for binding to TrkA or TrkB ECD (lower right panel). (C) ECD domain in TrkA is essential for amitriptyline to bind TrkA receptor. (D) Amitriptyline associates with the 1st LRM in the N-terminus of ECD of TrkA. Protein sequence alignment between the LRM motif from TrkA and the counterparts from TrkB and TrkC (upper panel). In vitro binding assay was conducted with a variety of fragments of TrkA ECD recombinant proteins (lower panel). (E) Amitriptyline provokes TrkA dimerization. GFP-TrkA and HA-TrkA or HA-TrkB were respectively cotransfected into HEK293 cells, and treated with 0.5 μM amitriptyline or imipramine for 30 min. GFP-TrkA was immunoprecipitated with anti-GFP antibody, the coprecipitated proteins were analyzed with anti-HA antibody. (F) Amitriptyline triggered TrkA or TrkB tyrosine phosphorylation in HEK293 cell individually transfected by TrkA or TrkB, respectively. Kinase-dead TrkA displayed negligible tyrosine phosphorylation.

Figure 5
Figure 5. Amitriptyline activates TrkA and TrkB receptors and prevents Kainic acid-triggered neuronal apoptosis in mouse brain

(A) Amitriptyline but not imipramine activates TrkA and TrkB receptors in mouse brain. 2−3 month old C57BL/6 mice were intraperitoneally injected with 15 mg/kg amitriptyline and imipramine (20 mg/kg) for various time points. Immunoblotting was conducted with various indicated antibodies. (B) Amitriptyline and imipramine do not alter TrkA or TrkB transcription. RT-PCR analysis of TrkA and TrkB in mouse brain. (C) Amitriptyline diminishes kainic acid-triggered hippocampal neuronal cell death. 2−3 month old C57BL/6 mice were intraperitoneally injected with 15 mg/kg amitriptyline either before or after kainic acid (25 mg/kg) administration. In 5 days, the brain slides were analyzed with TUNEL assay. Green stands for apoptotic nuclei, which were also stained with DAPI. (D) Amitriptyline increases TrkA expression and provokes TrkA and TrkB activation in hippocampus. Data represent mean ± S.E.M. (n = 3/group, Oneway ANOVA, Bonferroni post hoc test, *p<0.01; **p<0.01).

Figure 6
Figure 6. Amitriptyline promotes TrkA and TrkB hetero-dimerization in mouse brain

(A) Amitriptyline provokes TrkA phosphorylation and upregulates its expression mice. 2−3 months old mice were intraperitoneally injected with various antidepressant drugs for 5 days. The doses were: 15 mg/kg, 20 mg/kg and 25 mg/kg for amitriptyline, imipramine and fluoxetine, respectively. (B) Amitriptyline triggers TrkA and TrkB receptor heterodimerization in mouse brain. Wild-type and 5-HT1a null mice were treated with various antidepressant drugs for 5 days. TrkA was immunoprecipitated, and its coprecipitated proteins were analyzed by immunoblotting. (C) Amitriptyline elicits TrkA and TrkB receptor homo- and hetero-dimerization. Differentially tagged TrkA and TrkB receptors were cotransfected into HEK293 cells, and treated with various antidepressant drugs for 30 min. Data represent mean ± S.E.M of n = 3. (D) Amitriptyline binding motif on TrkA is essential for Trk receptor homo-, hetero-dimerization. HA-tagged TrkA or TrkB was cotransfected with GST-ΔTrkA or GST-ΔTrkB into HEK293 cells, followed by NGF, BDNF, amitriptyline or imipramine treatment. Truncated TrkA or TrkB was pulled down by glutathione beads, and monitored by anti-HA antibody.

Figure 7
Figure 7. Amitriptyline prevents neurons from apoptosis in a TrkA-dependent manner

(A) Amitriptyline activates TrkB independent of TrkA receptor. Cortical neurons (TrkA −/−) were pretreated with a variety of compounds for 30 min, the cell lysates were analyzed by immunoblotting with anti-p-TrkB, anti-p-TrkA. (B) Amitriptyline activates TrkA in TrkB-null neurons. Cortical neurons from TrkB +/− × TrkB +/− mice were treated with amitriptyline or imipramine for 30 min. Amitriptyline but not imipramine activated TrkA receptor in both wild-type and TrkB knockout neurons (top and 3rd panels). (C) Amitriptyline selectively activates TrkA F592A, which can be blocked by 1NMPP1. The primary cultures were pretreated for 30 min with either K252a (100 nM) or 1NMPP1 (100 nM), followed by 0.5 μM amitriptyline, imipramine or fluoxetine for 30 min. Amitriptyline-provoked TrkA activation was selectively inhibited by 1NMPP1 but not by K252a (top panel). (D) Amitriptyline suppresses KA-induced neuronal cell death in TrkA F592A mutant mice, which can be blocked by 1NMPP1. TrkA F592A knockin mice were treated with following reagents: saline, 1NMPP1, kainic acid, 1NMPP1 + kainic acid, amitriptyline, 1NMPP1 + amitriptyline, 1NMPP1 + amitriptyline + kainic acid, as described in the experimental section. Immunoblotting was conducted with the indicated antibodies. (E) TrkB activation is dispensable for the neuroprotective effect of amitriptyline.

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