Activated CRHR1 triggers Akt phosphorylation in hippocampal neuronal cells.

DOS SANTOS CLARO, P. A.; SILBERSTEIN, S.; ARMANDO, N.G.

Congreso; Cell Bio Virtual 2020, ASCB/EMBO meeting.; 2020

ASCB/EMBO

Corticotrophin releasing hormone (CRH) plays a key role in the regulation of neuroendocrine, autonomic, and behavioral response to stress. Dysregulation of the CRH/CRHR1 system is crucial in the pathogenesis of stress-related disorders. CRHR1 is a class B GPCR that signals mainly by Gs coupling, leading to an increase of cyclic AMP (cAMP) and the activation of multiple signaling cascades. Up to date the MAPKs pathway has been the most studied downstream of this receptor. We have previously described that CRHR1 activates G protein-dependent and receptor endocytosis-dependent mechanisms evidenced as a biphasic ERK1/2 activation in the mouse hippocampal cell line HT22 stably expressing CRHR1 (HT22-CRHR1). We have shown that upon stimulation, CRHR1 engages separate cAMP sources, involving the soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases. Both sources participate in the acute activation of ERK1/2, but only sAC activity is essential for the sustained phospho-ERK1/2 (pERK1/2) phase. Moreover, we demonstrated that CRHR1 continues to generate cAMP from endocytic compartments, uncovering a link between sAC and endosome based GPCR signaling. In the brain Akt activity mediates several cellular processes, such as neuronal survival and synaptic plasticity. PI3K-Akt signaling has been implicated in the etiology of mood disorders as well as in the antidepressant-like effects of various psychiatric drugs.In this work we wanted to assess whether the PI3K-Akt pathway was involved in CRH/CRHR1 signaling and if there was a crosstalk with the MAPKs cascade. In HT22-CRHR1 cells, Akt was phosphorylated downstream activated CRHR1 in a cAMP-dependent manner. Phosphorylated Akt (pAkt) was active as evidenced by the phosphorylation state of substrates. pAkt profile was opposite to that of pERK1/2 which suggested a possible crosstalk between these pathways. Inhibition of MEK had no effect on Akt activation, whilst pharmacological inhibition of either PI3K or Akt diminished pERK1/2 levels in response to CRH. On the contrary, PI3K-Akt inhibition appeared to increase CRH-mediated cAMP response and CREB phosphorylation. Given that Akt activation temporally overlapped with the endocytosis-dependent phase of ERK1/2 activation, we evaluated pAkt levels in cells where internalization had been blocked and found that CRHR1 endocytosis is indeed required for Akt phosphorylation.These results indicate that PI3K-Akt pathway is activated downstream CRHR1 in a neuronal context and that there is a crosstalk between Akt and ERK1/2. Moreover, Akt phosphorylation dependent on receptor internalization reinforces the idea of CRHR1 signaling from endosomes. Future work will involve the characterization of the endocytic compartments from which the receptor is signaling.