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Review
. 2020 Jun 24:11:417.
doi: 10.3389/fendo.2020.00417. eCollection 2020.

Anti-apoptotic Actions of Allopregnanolone and Ganaxolone Mediated Through Membrane Progesterone Receptors (PAQRs) in Neuronal Cells

Peter Thomas  1 , Yefei Pang  1
Affiliations
Review

Anti-apoptotic Actions of Allopregnanolone and Ganaxolone Mediated Through Membrane Progesterone Receptors (PAQRs) in Neuronal Cells

Peter Thomas et al. Front Endocrinol (Lausanne). .

Abstract

The neurosteroids progesterone and allopregnanolone regulate numerous neuroprotective functions in neural tissues including inhibition of epileptic seizures and cell death. Many of progesterone's actions are mediated through the nuclear progesterone receptor (PR), while allopregnanolone is widely considered to be devoid of hormonal activity and instead acts through modulation of GABA-A receptor activity. However, allopregnanolone can also exert hormonal actions in neuronal cells through binding and activating membrane progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor (PAQR) family. The distribution and functions of the five mPR subtypes (α, β, γ, δ, ε) in neural tissues are briefly reviewed. mPRδ has the highest binding affinity for allopregnanolone and is highly expressed throughout the human brain. Low concentrations (20 nM) of allopregnanolone act through mPRδ to stimulate G protein (Gs)-dependent signaling pathways resulting in reduced cell death and apoptosis in mPRδ-transfected cells. The 3-methylated synthetic analog of allopregnanolone, ganaxolone, is currently undergoing clinical trials as a promising GABA-A receptor-selective antiepileptic drug (AED). New data show that low concentrations (20 nM) of ganaxolone also activate mPRδ signaling and exert anti-apoptotic actions through this receptor. Preliminary evidence suggests that ganaxolone can also exert neuroprotective effects by activating inhibitory G protein (Gi)-dependent signaling through mPRα and/or mPRβ in neuronal cells. The results indicate that mPRs are likely intermediaries in multiple actions of natural and synthetic neurosteroids in the brain. Potential off-target effects of ganaxolone through activation of mPRs in patients receiving long-term treatment for epilepsy and other disorders should be considered and warrant further investigation.

Keywords: AED; PAQR agonists; allopregnanolone; ganaxolone; inhibition apoptosis; membrane progesterone receptors; neuronal cells; neuroprotective.

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Figures

Figure 1
Figure 1
Interactions of ganaxolone with human mPRδ (PAQR6). (A) Structures of ganaxolone and several other neurosteroids. (B) Representative competition curve of ganaxolone binding to plasma membranes of mPRδ-transfected MDA-MB-231 cells (231-mPRδ) expressed as percentage of maximum [3H]-progesterone binding. Ganaxolone was added to the assay buffer dissolved in ethanol. Ethanol was 0.1% of total volume, which did not affect [3H]-progesterone binding. P4, progesterone. (C,E,F) Effects of 4 days treatment with progesterone (P4), allopregnanolone (Allo) and ganaxolone (Gana) on serum starvation-induced percent apoptotic cells detected with a TUNEL assay kit (C) and percent cell death detected by trypan blue staining (E,F) of the vector-transfected MDA MB-231 cells and 231-mPRδ cells, N = 3. (D) Effect of treatment with ganaxolone (10 nM) for 15 min. on cellular cAMP levels in 231-mPRδ cells. N = 3. (G,H) Representative Western blot analysis and quantification of effects of 20-min treatments with progesterone (P), allopregnanolone (A), and ganaxolone (G) on activation of ERK. P-ERK: phosphorylated ERK, ERK: total ERK in the vector- (CTL cells), and 231-mPRδ cells. E100: 100 nM EGF as a positive control. The bar graph shows relative densitometry changes of the bands in Western blot images (N = 3). Results were analyzed by one-way ANOVA, followed by Newman-Keul's multiple comparison test. Treatment groups that are significantly different from each other in the post hoc test (P < 0.05) are indicated by different letters. Experiments were repeated three or more times, and similar results and similar significant differences between treatment groups were obtained on each occasion. See Pang et al. (15) for descriptions of reagents, culture conditions and assay procedures.
Figure 2
Figure 2
Effects of ganaxolone on rodent neuronal cell lines. (A) Effects of 4 days treatment with 20 nM and 100 nM progesterone (P4), allopregnanolone (Allo) and ganaxolone (Gana) on cell death of mouse hypothalamic GT1-7 cells. N = 3. (B) Detection of mPRα (α), mPRβ (β), mPRδ (δ), mPRε (ε), and progesterone receptor membrane component 1, PGRMC1 (PG) mRNA expression by RT-PCR in immortalized rat hippocampal H19-7 cells. (C) Representative Western blot analysis of effects of pre-incubation with muscimol (Musc, 100 μM) and bicuculline (Bicu, 1 μM) for 20 min on neurosteroid-induced (100 nM, for 20 min.) activation of ERK. P-ERK, phosphorylated ERK; ERK, total ERK in H19-7 cells; V, vehicle control; A, allopregnanolone; G, ganaxolone; P, progesterone. The bar graph shows relative densitometry changes of the bands in Western blot images (N = 3). (D) Effects of 15 min. treatments with 20 nM progesterone (P4), Org OD 02-0 (02), allopregnanolone (Allo) and ganaxolone (G, 20 and 100 nM) on cAMP levels in H19-7 cells. (N = 3). Results were analyzed by one-way ANOVA, followed by Newman-Keul's multiple comparison test. Treatment groups that are significantly different from each other in the post hoc test (P < 0.05) are indicated by different letters. Experiments were repeated three or more times, and similar results and similar significant differences between treatment groups were obtained on each occasion. See Pang et al. (15) for descriptions of reagents, culture conditions, and assay procedures.
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