Evidence for effective structure‐based neuromodulatory effects of new analogues of neurosteroid allopregnanolone

Abstract

The neurosteroid allopregnanolone (AP) modulates neuroendocrine/neurobiological processes, including hypothalamic-pituitary-adrenocortical activities, pain, anxiety, neurogenesis and neuroprotection. These observations raised the hope of developing AP-based therapies against neuroendocrine and/or neurodegenerative disorders. However, the pleiotropic actions of AP, particularly its cell-proliferation-promoting effects, hamper the development of selective/targeted therapies. For example, although AP-induced neurogenesis may serve to compensate neuronal loss in degenerative brains, AP-evoked cell-proliferation is contraindicated for steroid-sensitive cancer patients. To foster progress, we synthesised 4 novel AP analogues of neurosteroids (ANS) designated BR053 (12-oxo-epi-AP), BR297 (O-allyl-epi-AP), BR351 (O-allyl-AP) and BR338 (12-oxo-AP). First, because AP is well-known as allosteric modulator of GABAA receptors (GABAA-R), we used the electrophysiological patch-clamp technique to determine the structure-activity relationship of our ANS on GABAA-activated current in NCB20 cells expressing functional GABAA-R. We found that the addition of 12-oxo-group did not significantly change the respective positive or negative allosteric effects of 3α-AP or 3β-(epi)-AP analogues. Importantly, substitution of the 3α-hydroxyl-group by 3α-O-allyl highly modified the ANS activities. Unlike AP, BR351 induced a long-lasting desensitisation/inhibition of GABAA-R. Interestingly, replacement of the 3β-hydroxyl by 3β-O-allyl (BR297) completely reversed the activity from negative to positive allosteric action. In a second step, we compared the actions of AP and ANS on SH-SY5Y neuronal cell viability/proliferation using MTT-reduction assays. Different dose-response curves were demonstrated for AP and the ANS. By contrast to AP, BR297 was totally devoid of cell-proliferative effect. Finally, we compared AP and ANS abilities to protect against oxidative stress-induced neuronal death pivotally involved in neurodegenerative diseases. Both BR351 and BR297 had notable advantages over AP in protecting SH-SY5Y cells against oxidative stress-induced death. Thus, BR297 appears to be a potent neuroprotective compound devoid of cell-proliferative activity. Altogether, our results suggest promising perspectives for the development of neurosteroid-based selective and effective strategies against neuroendocrine and/or neurodegenerative disorders.

RETRACTED: Allopregnanolone Modulates GABAAR-Dependent CaMKIIδ3 and BDNF to Protect SH-SY5Y Cells Against 6-OHDA-Induced Damage

“…Moreover, the action of APα has been shown to activate GABAAR-driven VGLCC and subsequent increases of intracellular Ca 2+ concentration (Keller et al, 2004;Wang et al, , 2010Wang and Brinton, 2008;Jagasia et al, 2009;Chen S. et al, 2011;Frye et al, 2014), which was in accordance with our current study. In addition, we also cannot rule out the possibility that APα interacts with the pregnane-X-receptor or membrane progesterone G-protein coupled receptor to regulate its neuroprotective process (Charalampopoulos et al, 2008;Mendell et al, 2018;Taleb et al, 2018). Mendell et al (2018) also suggested a model for neurosteroid actions included both GABAAR-dependent and GABAAR-independent mechanisms.…”

“…In this study, all reagents, materials, and chemicals were analytical grade and highest purity, including human neuroblastoma SH-SY5Y cell lines (SCSP-5014, Chinese Academy of Sciences, China); Dulbecco's modified Eagle's medium (DMEM, 11965092), Neurobasal (21103049), B27 (17504044), fetal bovine serum (FBS), glutamax (35050061), trypsin-EDTA (25300062) and sodium pyruvate (11360070; Gibco Invitrogen, MA, USA); APα (3653) and Bic (485494; Tocris, USA); Nifedipine (ab120135, Abcam, Burlingame, CA, USA); RA (R2500) and 6-OHDA (H4381; Sigma-Aldrich, St. Louis, MI, USA); penicillin and streptomycin (Solarbio, Beijing, China); bovine serum albumin (BSA) and BrdU (Bio-Sharp, China); DMSO (ST038), EGTA (ST068), phenylmethanesulfonyl fluoride (PMSF, ST506), TritonX-100, cytosolic and nuclear extraction kit (P0028; Beyotime, China). The concentration of key chemicals was chosen according to the relevant references (Liu et al, 2002;Miloso et al, 2004;Wang et al, , 2010Gilany et al, 2008;Wang and Brinton, 2008;Fernandes et al, 2017;Xie et al, 2017;Taleb et al, 2018). All experiments should comply with the standard biosecurity and safety procedures from the ethical committee of Wenzhou Medical University according to the National Institute of Health Guide for Care and Use of Laboratory Animals (NIH Publications No.…”

Microglial Cell Morphology and Phagocytic Activity Are Critically Regulated by the Neurosteroid Allopregnanolone: A Possible Role in Neuroprotection

“…Neurosteroid ALLO has been reported to induce the proliferation of different cell types [ 37 , 38 ]. This parameter is important in the context of autoimmune diseases, where an uncontrolled proliferation of immune cells could exacerbate the inflammation.…”

“…Steroid hormones are known to modulate cell proliferation. ALLO has also been reported to increase the proliferation of different cell types, such as postnatal hippocampal cells [ 37 ], SH-SY5Y cells [ 38 ], or Schwann cells [ 80 ]. Neither the microglial BV-2 nor the oligodendroglial 158N cell line used in this study showed a modified proliferation after the administration of ALLO in the range of 10 nM to 10 µM ( Figure 3 C,D or Figure 6 C).…”

New perspectives for the action of steroids in the brain

“…The last paper in this section is an experimental study describing the properties of BR297 (a new analogue of allopregnanolone) that appears to be a potent neuroprotective compound devoid of cell‐proliferative activity. These results open promising perspectives for the development of neurosteroid‐based selective and effective strategies against neuroendocrine and/or neurodegenerative disorders …”