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

DURAND DANIELA; CARNIGLIA LILA; CARUSO CARLA; LASAGA MERCEDES

Taormina

Congreso; 7th International meeting on metabotropic glutamate receptors; 2011

Universidad de Roma "Sapienza"

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 [1]. 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. We showed by the MTT method that both a non-hydrolyzable analog of cAMP, dibutyryl-cAMP (1 mM), and an Akt1/2 inhibitor (0.5 uM) abolished the anti-apoptotic effect of the mGluR3 agonists LY379268 or LY404039 (100 uM) on cultured astrocytes exposed to 1 mM NO (p0.001 vs NO+LY). However, a cGMP analog, 8Br-cGMP (1 mM), did not modify the effect of LY379268 on cell viability. These data indicate that the mGluR3-induced Akt activation and reduction in cAMP levels are responsible for the protective actions of these receptors in astrocytes. Moreover, LY404039 per se induced Akt phosphorylation (p0.05 vs control), whereas phosphorylated Akt levels were diminished by NO (p0.001 vs control), an effect that was absent in the presence of LY404039. Further, dibutyryl-cAMP impairs Akt phosphorylation induced by LY404039 (p0.01 vs LY404039), indicating a relationship between mGluR3-reduced cAMP levels and PI3K/Akt pathway activation. NF-kB family proteins mediate inflammatory responses and survival signaling. Opposite to p65-p50, c-Rel containing dimers promote transcription of anti-apoptotic genes [2]. Thus, we determined p65-c-Rel levels in nuclear extracts by co-immunoprecipitation followed by Western-blot. mGluR3 agonists not only induce per se survival-linked p65-c-Rel dimers (p0.05 vs control), but also impede reduction of levels of p65-c-Rel dimers caused by NO (p0.05 vs NO), without altering nuclear levels of free p65 nor c-Rel, 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. [1] Durand et al., 2010, J. Neurochem., 112, 420-433. [2] Sarnico et al., 2009, J. Neurochem., 108, 475-485.