Reduced cAMP, Akt activation and p65-c-Rel dimerization: mechanisms involved in the protective effects of mGluR3 agonists in cultured astrocytes

Abstract

In recent decades, astrocytes have emerged as key pieces in the maintenance of normal functioning of the central nervous system. Any impairment in astroglial function can ultimately lead to generalized disturbance in the brain, thus pharmacological targets associated with prevention of astrocyte death are actually promising. Subtype 3 of metabotropic glutamate receptors (mGluR3) is present in astrocytes, its activation exerting neuroprotective roles. In fact, we have previously demonstrated that mGluR3 selective agonists prevent nitric oxide (NO)-induced astrocyte death. However, mechanisms responsible for that cytoprotective property are still subject to study. Although inhibition of adenylyl cyclase by mGluR3 activation was extensively reported, the involvement of reduced cAMP levels in the effects of mGluR3 agonists and the association between cAMP decrease and the downstream pathways activated by mGluR3 remain neglected. Thus, we studied intracellular signaling mediating anti-apoptotic actions of mGluR3 in cultured rat astrocytes exposed to NO. In the present work, we showed that the cytoprotective effect of mGluR3 agonists (LY379268 and LY404039) requires both the reduction of intracellular cAMP levels and activation of Akt, as assessed by MTT and TUNEL techniques. Moreover, dibutyryl-cAMP impairs Akt phosphorylation induced by LY404039, indicating a relationship between mGluR3-reduced cAMP levels and PI3K/Akt pathway activation. We also demonstrated, by co-immunoprecipitation followed by western-blot, that the mGluR3 agonists not only induce per se survival-linked interaction between members of the NF-κB family p65 and c-Rel, but also impede reduction of levels of p65-c-Rel dimers caused by NO, suggesting a possible anti-apoptotic role for p65-c-Rel. All together, these data suggest that mGluR3 agonists may regulate cAMP/Akt/p65-c-Rel pathway, which would contribute to the protective effect of mGluR3 against NO challenge in astrocytes. Our results widen the knowledge about mechanisms of action of mGluR3, potential targets for the treatment of neurodegenerative disorders where a pathophysiological role for NO has been established.

Figure 1. mGluR3 expression in astrocytes.

Immunolabeling for mGluR2/3 (green), GFAP (red) and DAPI (blue) done on rat primary astrocytes. (A) Immunolabeling for mGluR2/3. Picture B shows mGluR2/3 and GFAP merged images (yellow).

Figure 2. Expression of NF-κB family proteins.

Astrocyte primary cultures were exposed to LPS/IFN-γ (1 µg/mL/50 ng/mL)±100 µM LY379268 for 30 min and nuclear and cytosolic proteic extracts were used for western-blots of p65 (A), IκBα (B) and c-Rel (C). Values of OD from bands were normalized to the internal control β-actin and data expressed relative to control group. Bars represent the mean ± SEM of 5–6 (A and B) or 3 (C) independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus control. (D) The purity of nuclear and cytosolic fractions was assayed by western-blot for nuclear histone.

Figure 3. Both LPS/IFN-γ and LY379268 induce p65-c-Rel dimerization.

Astrocytes were incubated with LPS/IFN-γ (1 µg/mL/50 ng/mL)±100 µM LY379268 for 30 min. Interaction between p65 and c-Rel proteins was analyzed from 70 µg of proteic nuclear extracts by co-immunoprecipitation (IP) with anti-c-Rel antibody followed by western-blot (WB) for p65. (A) Specificity of the assay was analyzed by immunoprecipitating nuclear proteins with anti-c-Rel antibody (lane 1) or with an irrelevant IgG of the same origin as the anti-c-Rel antibody (lane 2), which was not able to precipitate c-Rel-linked p65. (B) Levels of anti-c-Rel-immunoprecipitated p65 were expressed relative to control group. Bars represent the mean ± SEM of 3 independent experiments. *p<0.05, **p<0.01 versus control. Input corresponds to total content of p65 in nuclear extracts.

Figure 4. mGluR3 activation avoids inhibition of p65-c-Rel dimerization provoked by DETA/NO.

p65-c-Rel interaction was analyzed by co-immunoprecipitation (IP) followed by western-blot (WB) of nuclear extracts from astrocytes treated with 1 mM DETA/NO±100 µM LY404039 for 30 min. Levels of anti-c-Rel-immunoprecipitated p65 were expressed relative to control group. Bars represent the mean ± SEM of 3 independent experiments. *p<0.05 versus control, ∧p<0.05 versus DETA/NO. Input corresponds to total content of p65 in nuclear extracts.

Figure 5. Cytoprotective action of mGluR3 agonists is mediated by reduction of cAMP levels but not by cGMP.

Astrocyte primary cultures were incubated with 1 mM DETA/NO in the presence or absence of 100 µM LY379268 and/or 1 mM db-cAMP or 1 mM 8Br-cGMP for 48 h. Cell viability was assayed by the MTT method. Bars represent the mean ± SEM of 6–8 determinations per group, of one representative experiment of 2 independent ones. *p<0.05 versus control; ∧p<0.05 versus DETA/NO; ###p<0.001 versus DETA/NO+LY379268.

Figure 6. Akt activity mediates the antiapoptotic effect of mGluR3 agonists in astrocytes.

(A) A dose-response curve was plotted by a MTT assay to determine the optimal concentration of the Akt inhibitor. (B) Astrocytes were treated with 1 mM DETA/NO in the presence or absence of 100 µM LY404039 and/or Akt inhibitor (0.5 µM) for 48 h. Cell viability was assayed by the MTT method. Bars represent the mean ± SEM of 6–8 determinations per group of one representative experiment of 2 independent ones. **p<0.01 versus control; ∧∧∧p<0.001 versus DETA/NO; ###p<0.001 versus DETA/NO+LY404039.

Figure 7. LY404039 avoids reduction in phospho-Akt levels caused by DETA/NO.

Total cellular proteins were extracted as described in Materials and Methods from astrocytes exposed to 1 mM DETA/NO±100 µM LY404039±1 mM db-cAMP for 30 min, and 50 µg of protein extracts were assayed for phospho-Akt by western-blot. (A) Specificity of the western-blot was analyzed by incubating astrocytes with the PI3K/Akt pathway inhibitor, 20 µM LY294002, which completely prevented LY404039-induced Akt phosphorylation, and LPS/IFN-γ was used as positive control of Akt activation. Membranes were stripped and incubated with anti-total Akt antibody, which was considered loading control. (B) Data were expressed as phospho-Akt/total Akt ratio and related to control group. Bars represent the mean ± SEM of 4 independent experiments. *p<0.05, ***p<0.001 versus control, ∧∧p<0.01 versus LY404039.

Figure 8. Proposed model for mGluR3 protective actions in astrocytes.

mGluR3 activation by selective ligands couples to Gi protein activation, which can inhibit adenylyl cyclase activity and cAMP production and/or directly activate PI3K/Akt pathway. Alternatively, cAMP reduction could lead to Akt activation, because cAMP inhibits Akt phosphorylation. In turn, Akt might induce p65-c-Rel interaction at nucleus, which stimulates the transcription of anti-apoptotic genes such as Bcl-2, resulting in astrocyte protection from NO challenge.