Activation of Trk neurotrophin receptors in the absence of neurotrophins

Abstract

Neurotrophins regulate neuronal cell survival and synaptic plasticity through activation of Trk receptor tyrosine kinases. Binding of neurotrophins to Trk receptors results in receptor autophosphorylation and downstream phosphorylation cascades. Here, we describe an approach to use small molecule agonists to transactivate Trk neurotrophin receptors. Activation of TrkA receptors in PC12 cells and TrkB in hippocampal neurons was observed after treatment with adenosine, a neuromodulator that acts through G protein-coupled receptors. These effects were reproduced by using the adenosine agonist CGS 21680 and were counteracted with the antagonist ZM 241385, indicating that this transactivation event by adenosine involves adenosine 2A receptors. The increase in Trk activity could be inhibited by the use of the Src family-specific inhibitor, PP1, or K252a, an inhibitor of Trk receptors. In contrast to other G protein-coupled receptor transactivation events, adenosine used Trk receptor signaling with a longer time course. Moreover, adenosine activated phosphatidylinositol 3-kinase/Akt through a Trk-dependent mechanism that resulted in increased cell survival after nerve growth factor or brain-derived neurotrophic factor withdrawal. Therefore, adenosine acting through the A(2A) receptors exerts a trophic effect through the engagement of Trk receptors. These results provide an explanation for neuroprotective actions of adenosine through a unique signaling mechanism and raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of neurodegenerative diseases.

Figure 1

Activation of TrkA receptors with G protein-coupled receptor ligands. Stably transfected PC12 cells expressing high levels of TrkA (615) were treated with the indicated compounds for 2 h. The cells were subsequently harvested in lysis buffer as described in Materials and Methods. Lysates were immunoprecipitated with anti-pan Trk rabbit antiserum. Immunocomplexes were analyzed by immunoblotting with anti-phosphotyrosine antibody (PY99). Immunoprecipitation of TrkA receptors was then confirmed by immunoblotting of the immunocomplex with anti-pan Trk antiserum.

Figure 2

Time course and dose of adenosine activation of TrkA receptors. (A) Different concentrations of adenosine were administered to PC12 615 cells for 2 h or NGF (1 ng/ml) for 10 min. Cells were also treated with CGS 21680 at the indicated doses for 2 h. (B) PC12 cells (615) were treated with adenosine (10 μM) for various times or with 5 ng/ml NGF for 10 min. Phosphorylated TrkA receptors were detected by immunoblot analysis using PY99 anti-phosphotyrosine antibodies. The amount of Trk receptors in each condition was verified by immunoblotting.

Figure 3

Adenosine activation of TrkA by A_2A receptors. (A) PC12 cells (615) were treated with the A_2A agonist CGS 21680 (10 nM) and an A₁ agonist CPA (10 nM) for 2 h. ZM 241385 (10 nM), an A_2A antagonist, was incubated with the cells for 15 min before treatment with adenosine (10 μM) for 2 h. (B) PC12 cells (615) were incubated with the indicated concentrations of PP1, a Src family kinase inhibitor (30), for 30 min, and then treated with adenosine (10 μM) for 2 h. Activation of TrkA was assessed by immunoprecipitation and immunoblot analysis using PY99 anti-phosphotyrosine antibody.

Figure 4

Adenosine activation of TrkB receptors in hippocampal neurons. Primary cultures of E17 hippocampal neurons were prepared as described in Materials and Methods and treated with (A) CGS 21680 (10 nM) or BDNF (1 ng/ml) for various times and (B) adenosine (10 μM), CGS 21680 (10 nM), CPA (10 nM), or BDNF (10 ng/ml) for 2 h. Activation of TrkB receptors was assessed by immunoprecipitation and Western blotting with anti-phosphotyrosine antibody.

Figure 5

Effects of adenosine on MAP kinase and Akt activation. PC12 cells (615) were treated with adenosine (10 μM) for various times in the presence or absence of K252a (100 nM) or LY294002 (10 μM). The cells were subsequently harvested in lysis buffer; lysates and immunoprecipitated samples were subsequently immunoblotted with anti-phospho-MAP kinase, anti-phospho-Akt, and anti-phosphotyrosine. Reprobing with anti-MAP kinase and anti-pan-Trk antibodies was carried out to ensure equal protein loading.

Figure 6

Trophic effects of adenosine agonist in PC12 and hippocampal cells deprived of neurotrophins. (A) NGF-differentiated PC12 cells were prepared, and then NGF and serum were withdrawn for 48 h as described in Materials and Methods. On NGF withdrawal, various concentrations of CGS 21680 (CGS) were added to the media. CON, no addition. NGF (50 ng/ml), insulin-like growth factor 1 (=IGF-1) at 100 ng/ml, and CGS 21680 (10 nM) were added together with K252a (100 nM), LY294002 (10 μM), or PD98059 (25 μM) upon NGF withdrawal. (B) Hippocampal neurons were prepared and B27 was withdrawn for 48 h as described in Materials and Methods. Upon B27 withdrawal, various concentrations of CGS 21680 (CGS) were added to the media. CGS 21680 (10 nM) and BDNF (100 ng/ml) were added together with K252a (100 nM) on B27 withdrawal. All LDH levels were quantitated and % cell death calculated as described in Materials and Methods. All bars depict mean + SEM from three independent experiments.