Abstracts

Rationale: Neuroactive steroids (NAS) are progesterone derivatives that exert potent anticonvulsant, anxiolytic, and sedative properties. Recent FDA approval of the synthetic NAS Ganaxolone to arrest refractory seizures in cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder and of Brexanolone to treat postpartum depression has increased interest in NAS as novel therapeutic treatments. While primarily studied as positive allosteric modulators (PAMs) of the γ-aminobutyric type A receptor (GABA_AR), prior research has demonstrated NAS also modulate GABAergic inhibition via non-allosteric mechanisms involving membrane progesterone receptors (mPRs). mPRs are a class of G-protein coupled receptors consisting of five subtypes (mPRα, mPRβ, mPRγ, mPRδ, and mPRε). However, the role of mPRs in the brain has not been addressed. Therefore, we aimed to investigate the non-allosteric effects of NAS on GABA_ARs via mPRs by characterizing intracellular signaling pathways that converge on kinases known to modulate GABA_AR phosphorylation and trafficking.

Methods: To investigate mPR signaling, we expressed each mPR in isolation in MDA231 cells (a triple negative breast cancer cell line that does not express endogenous progesterone receptors). Following lentiviral transduction with a mPR-GFP construct, cells were acutely exposed to a pan-mPR agonist, ORG, that does not act on GABA_ARs, or the endogenous NAS, allopregnanolone (ALLO), a GABA_AR PAM. Kinase activation was assessed with immunoblotting and the potency of these compounds for each mPR was calculated via G_s and G_q luminescent reporter assays. Finally, acute hippocampal brain slices from adult male and female wild-type (WT) C57Bl/6 mice were treated with NAS or ORG to assess for significance of our results in the MDA231 cell lines.


Results: In MDA231-mPRδ cells, NAS increased PKC activity via G_q activation at a high potency with an EC₅₀ of around 10nM. This contrasted signaling in MDA231-mPRε cells where NAS increased PKA activity via G_s activation with a similar potency. mPRδ and mPRε also displayed differences in internalization, where only mPRδ demonstrated rapid agonist induced internalization. In the forebrain of WT mice, mPRδ expression was 7-fold higher in females compared to males, while mPRε expression was 0.7-fold higher in females. Consistent with this greater sex-specific difference in mPRδ expression, only female WT mice displayed significant activation of PKC following NAS or ORG treatment.


Conclusions: Collectively, these results highlight the first evidence that mPRδ and mPRε display divergent signaling via G_q and G_s, respectively, and that mPR activation leads to a sex-specific effect on intracellular signaling in the brain. Additionally, the high potency of NAS for mPRs is at concentrations similar to endogenous ALLO in the brain and is below demonstrated plasma levels of FDA approved NAS treatments. Therefore, this work emphasizes the importance of understanding mPR signaling in the brain and the ability of mPRs to modulate neuronal inhibition to better develop future novel therapies.


Funding: NIMH R01MH118263, NINDS R01NS108378, and Sage Therapeutics