Activation of Trk neurotrophin receptors by glucocorticoids provides a neuroprotective effect

Jeanneteau et al. 10.1073/pnas.0709102105.

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Fig. 6. Specificity of phospho-TrkB antibodies in primary neurons. We designed two distinct rabbit polyclonal anti-phosphopeptide antibodies to discriminate between TrkA and TrkB phosphorylation at the PLCg-binding site. The phospho-TrkA antibodies recognize the residue Y794-P whereas the phospho-TrkB antibodies recognize the residue Y816-P, both being the binding sites for PLCg. Primary cultures of embryonic E18 septal neurons were treated with NGF (50 ng.ml-1, 10 min), and hippocampal neurons were treated with BDNF or NT3 (50 ng.ml-1, 10 min). Expression of TrkA, TrkB and pan-Trk was determined by using the respective antibodies RTA, TrkB and C14.





Fig. 7. Corticosterone levels in P18 rats. Trunk blood was collected in EDTA-coated tubes. Plasma corticosterone levels were measured by ELISA. Corticosterone levels were elevated 9 h postadministration of metyrapone (IP, 150 mg.kg-1) due to the inhibition of the negative feedback of the hypothalamo-pituitary-adrenal (HPA) axis. Co-administration of dex (IP, 10 mg.kg-1) 3 h after metyrapone reduced plasma corticosterone levels due to the activation of the negative feedback of the HPA axis. P18 females show stronger amplitude variations of their corticosterone level than males after metyrapone and dexamethasone treatments.





Fig. 8. Phospho-TrkB immunoreactivity in hippocampus subfields. P18 rats were administered vehicle (IP, 0.9% saline) or dex (IP, 10 mg.kg-1) for 6 h. Arrows indicates p-TrkB immunoreactivity in the GCL and SGZ of the dentate gyrus. Counterstaining with the NeuN marker indicates only neuronal cells.





Fig. 9. Correlation between TrkB and GR phosphorylation in the striatum. Postnatal (P18-20) rats were administered i.p. dexamethasone-21 sodium salts (10 mg.kg-1) or PBS for 6 h. Dorsal striatum was carefully dissected and processed for biochemical detection of phospho-TrkB, TrkB, and Phospho-GR (S211-P), GR. There is a significant increase of phospho-TrkB in the striatal but not septal lysates of dex- compared to vehicle-treated animals (mean ± SEM). Normalization of phospho-TrkB/ TrkB ratio with phospho-GR/ GR ratio also showed that TrkB phosphorylation is correlated with GR phosphorylation in the striatum. Phospho-TrkB immunoreactivity in the striatum of dex-treated animals was detected in the subventricular zone (SVZ), an established site of neurogenesis.





Fig. 10. Trophic effects of glucocorticoids in cultured cortical neurons. Upon B27-supplement withdrawal for 72 h, primary neurons rapidly died as monitored by counting the number of apoptotic nuclei (2000 cells per conditions). Treatments: BDNF (50 ng.ml-1), dexamethasone (indicated concentrations) or vehicle (EtOH, 0.03%). Trk-mediated cell survival was abolished by LY29002 (10 mM) or mifepristone (5 mM), and reduced significantly by K252a (100 nM) or spironolactone (5 mM). % cell survival is expressed as mean ± SEM and the experiment was repeated 4 times. * P < 0.05, *** P < 0.001.





Fig. 11. Effect of transcription and translation inhibitors upon GC-induced Trk phosphorylation. Pretreatment of PC12-TrkA cells with actinomycin D (Act.D, 1 mg.ml-1) or cycloheximide (CHX, 50 mg.ml-1) prevented TrkA phosphorylation in response to 1 mM corticosterone for 3 h. Levels of phospho-TrkA were detected from Trk immunopecipitates.





Fig. 12. Effect of GCs on TrkB expression using a TrkB luciferase-promoter assay. TrkB is under the control of two promoters, called P1 and P2, the latter being sensitive to depolarization. Cortical neurons were transfected with two plasmids to co-express renilla with luciferase-P1, -P2 or empty vector (pGL3-basic). Treatments: KCL (30 mM, 5 h), Dex (1 mM, 5 h), mifepristone (1 mM, 5 h) serves to normalize fluorescence. Luciferase activity was normalized to Renilla fluorescence. * P < 0.05, n = 3.

This Article

  1. Published online before print March 17, 2008, doi: 10.1073/pnas.0709102105 PNAS March 25, 2008 vol. 105 no. 12 4862-4867
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