Studies on G-protein coupled receptors that are activated by neuroactive steroids

Abstract. Neuroactive steroids (NAS) such as allopregnanolone (ALLO) are synthesized in the brain from progesterone (P4) and exert potent anxiolytic, anticonvulsant, antidepressant, and hypnotic effects on behavior. Consistent with this, NAS have been approved as the first treatments to alleviate postpartum depression. NAS

are believed to exert their efficacy as positive allosteric modulators (PAM) of GABAARs, leading to acute elevations in the magnitude of phasic and tonic inhibition. In addition to this, we have demonstrated that NAS promote the insertion of GABAARs into the neuronal plasma membrane, leading to selective and sustained

elevations in tonic inhibition. Likewise, NAS promote cell survival and tubulin stability independent of their efficacy as GABAAR PAMs. One explanation for these pleiotropic effects is that they are mediated by specific NAS receptors. Consistent with this, studies in yeast, fish and mammalian cell lines have demonstrated that P4

and NAS bind to a family of putative membrane progesterone receptors (mPRs) with 5 members - ɑ, β, γ, δ and ε - that share homology with classic serpentine G-protein coupled receptors (GPCRs). Intriguingly, some mPR isoforms are highly expressed in the brain, suggesting they maybe of significance as effectors for NAS; however,

it remains to be established whether mPRs are bona fide metabotropic receptors for NAS. Likewise, their role in mediating the effects of NAS neuronal excitability has yet to be evaluated due to a lack of suitable pharmacological tools. Here we will examine the role that mPRs play in mediating NAS signaling in the DG a

domain of the hippocampus that predominantly expresses mPRɑ , and δ with lower levels of ε. To do so we will first assess whether exposure to NAS of clonal cell lines expressing these mPRs modulates cAMP signaling and PKC activity, accepted effectors for GPCRs. Using mice in which the expression of individual mPR isoforms has

been ablated using conditional and constitutive gene editing, we will evaluate their roles in mediating the effects of NAS on neuronal excitability. Preliminary studies have allowed us to formulate an overarching hypothesis; NAS exert metabotropic effects on the magnitude of tonic inhibition of mPRδ and mPRɑ, which in turn

signal via distinct effectors to increase and decrease the magnitude of tonic inhibition respectively, a process that mediates the sustained effects of NAS on neuronal excitability. Our study will focus on the following aims. Aim 1. To test the hypothesis that NAS exert pleiotropic effects on intracellular signaling via the

activation of distinct mPRs. Aim 2. To test the hypothesis that mPRδ selectively contributes to metabotropic effects of NAS on tonic inhibition. Aim 3. To test the hypothesis that chronic activation of mPRδ increases tonic inhibition and modifies the subunit composition of extrasynaptic GABAARs. Aim 4. To test the hypothesis that

NAS limit the magnitude of tonic currents via activation of mPRɑ. Collectively our study will provide the first insights into how NAS induces long-lasting effects on neuronal excitability, information that may ultimately aid the development of new strategies to improve mental health.