Adenosine receptor A2A-R contributes to motoneuron survival by transactivating the tyrosine kinase receptor TrkB

Abstract

Neurotrophins are potent survival factors for developing and injured neurons. However, they are not being used to treat neurodegenerative diseases because of difficulties in administration and numerous side effects that have been encountered in previous clinical trials. Their biological activities use Trk (tropomyosin-related kinase) transmembrane tyrosine kinases. Therefore, one alternative approach is to use transactivation pathways such as adenosine 2A receptor agonists, which can activate Trk receptor signaling independent of neurotrophin binding. However, the relevance in vivo and applicability of these transactivation events during neurodegenerative and injury conditions have never been extensively studied. Here we demonstrate that motoneuron survival after facial nerve lesioning is significantly enhanced by transactivation of Trk receptor tyrosine kinases by adenosine agonists. Moreover, survival of motoneurons directly required the activation of the BDNF receptor TrkB and an increase in Akt (AKT8 virus oncogene cellular homolog) activity. The ability of small molecules to activate a trophic response by using Trk signaling provides a unique mechanism to promote survival signals in motoneurons and suggests new strategies for using transactivation in neurodegenerative diseases.

Fig. 1.

The A_2A-R agonist CGS21680 rescues motoneurons from cell death after facial nerve lesioning. Survival of lesioned facial motoneurons of 1-day-old mice is enhanced in the presence of the adenosine receptor agonist CGS21680. (a, b, c, and d) The facial nerve of 1-day-old mice was lesioned unilaterally, and CGS21680 (a), ZM241385 (b), BDNF (c), or buffer alone (d) was applied to the proximal nerve stump in gel foam. Mice were killed 7 days later, and motoneurons in the facial nucleus were counted in serial sections on both the lesioned side and the unlesioned control side. (e) Values show mean ± SEM of survival rates relative to the unlesioned control side in the individual groups (CGS group: n = 6; ZM group: n = 3; control group: n = 3; BDNF group: n = 4; BDNF + ZM group: n = 4). P < 0.05 (*, P = 0.02131; **, P = 0.00342; n.s, not significant).

Fig. 2.

The A_2A-R agonist CGS21680 activates the TrkB receptor after facial nerve lesioning. After unilateral facial nerve transection at postnatal day 1 and local application of CGS21680 to the lesion site, facial nuclei were microdissected 6 h later and processed for immunoprecipitation of TrkB. (a) The precipitates were subjected to Western blot analysis for TrkB and phosphotyrosine. Band intensities were measured by using the AIDA software. (b) The ratio of phosphotyrosine to TrkB was determined for each animal, and quantification and statistical analysis are shown. au, absorbance units. Results reflect mean ± SEM from three independent experiments with n ≥ 3 in each group. *, P < 0.05.

Fig. 3.

The A_2A-R agonist CGS21680 supports survival of isolated embryonic motoneurons from lumbar spinal cord of 12.5-day mouse embryos. Enriched motoneurons from lumbar spinal cord were treated with CGS21680 (CGS), the A_2A-R antagonist ZM241385 (ZM), and BDNF as a positive control. After 7 days in culture, surviving motoneurons were counted. Data shown represent the percentage (mean ± SEM) of survival of originally plated cells. (a) Treatment with BDNF and the A_2A-R agonist CGS21680 resulted in enhanced survival in comparison with cultures that were treated with CGS21680 resulted in enhanced survival in comparison with cultures that were treated with no factor. There was no additive effect on survival when BDNF and CGS21680 were added in combination to motoneuron cultures. (b) Motoneurons isolated from the lumbar spinal cord of 12.5-day trkB^+/− intercross single mouse embryos were treated with BDNF, CGS21680, or GDNF. Survival with CGS21680 or BDNF but not with GDNF is reduced to background levels in trkB^−/− motoneurons. (c) When a neutralizing antiserum against BDNF was added at a dilution of 1:1,000 to the motoneuron cultures, the effect of BDNF was reduced to background levels. P < 0.05, (***, P = 0.00076). In contrast, the BDNF antibodies did not reduce the survival effect of CGS21680, indicating that BDNF is not mediating the effects of CGS21680 on survival of motoneurons via TrkB. Each experiment was performed at least three times, with n > 3 for each group. (d) Immunoprecipitation of Trk receptors from cultured motoneurons and detection of TrkB reveals that the BDNF antiserum blocks activation of TrkB when cultured motoneurons are stimulated with BDNF but does not block activation of TrkB in the presence of CGS21680.

Fig. 4.

Akt kinase is activated by the A_2A-R agonist CGS21680 in facial motoneurons lesioned on postnatal day 1. The brainstem containing the facial nuclei from the lesioned mice was prepared and processed for immunohistochemical detection of phospho-Ser-473-Akt. Staining is yellow in these micrographs. (a, b, and e) Akt is specifically phosphorylated when the facial nerve stumps were treated with CGS21680 (b) or BDNF (f). (a–f) Cells from the unlesioned control side corresponding to CGS21680-treated motoneurons (a), lesioned side containing CGS21680-treated motoneurons (b), unlesioned side corresponding to ZM241385-treated motoneurons (c), lesioned side containing the ZM241385-treated motoneurons (d), untreated controls (secondary antibody only) (e), and BDNF-treated motoneurons (f). (Scale bar: 20 μm.) Activation of Akt was enhanced after application of BDNF or CGS21689 and was reduced to low or undetectable levels when the nerve stump was treated with ZM241385 (d).