Neuroactive Steroids: Receptor Interactions and Responses

Neuroactive steroids (NASs) are naturally occurring steroids, which are synthesized centrally as de novo from cholesterol and are classified as pregnane, androstane, and sulfated neurosteroids (NSs). NASs modulate many processes via interacting with gamma-aminobutyric acid (GABA), N-methyl-d-aspartate, serotonin, voltage-gated calcium channels, voltage-dependent anion channels, α-adrenoreceptors, X-receptors of the liver, transient receptor potential channels, microtubule-associated protein 2, neurotrophin nerve growth factor, and σ1 receptors. Among these, NSs (especially allopregnanolone) have high potency and extensive GABA-A receptors and hence demonstrate anticonvulsant, anesthetic, central cytoprotectant, and baroreflex inhibitory effects. NSs are also involved in mood and learning via serotonin and anti-nociceptive activity via T-type voltage-gated Ca2+ channels. Moreover, they are modulators of mitochondrial function, synaptic plasticity, or regulators of apoptosis, which have a role in neuroprotective via voltage-dependent anion channels receptors. For proper functioning, NASs need to be in their normal level, whereas excess and deficiency may lead to abnormalities. When they are below the normal, NSs could have a part in development of depression, neuro-inflammation, multiple sclerosis, experimental autoimmune encephalitis, epilepsy, and schizophrenia. On the other hand, stress and attention deficit disorder could occur during excessive level. Overall, NASs are very important molecules with major neuropsychiatric activity.

testosterone, and progesterone-which are their blood-borne precursors (3). Moreover, they are also produced in fetoplacental unit (4). The term "neurosteroids" (NSs) are coined since cholesterol is the precursor for de novo synthesis of NSs centrally (5).
Dehydroepiandrosterone acts as an antagonist of cortisol and is the most plentiful circulating steroid among the NASs in human being (9). The sulfated form of this NASs-DHEA-S-has a relatively long half-life and in animal models, DHEA-S enhances cognitive and behavioral performance (10). Androstenol is a special type of NSs, acts as a pheromone, and has a higher structural resemblance to gamma-aminobutyric acid-A (GABA-A) receptor modulating NSs and as a result of this, act as a signaling molecule between entities of the alike species through interaction with GABA-A receptors (11).

BiOSYNTHeSiS OF NASs
Reduction of the A-ring from testosterone, deoxycorticosterone, and progesterone-which are steroid hormones-results in the formation of NSs (4). These chemicals can be released into the blood and act systemically or synthesized de novo locally, from cholesterol (5,18), in certain brain parts such as in the pineal gland (the major site for neurosteroidogenic organ), cortex, glutamatergic neurons, hippocampus, and cortex (19).
Allopregnanolone and 7α-OH PREG were exceedingly produced and show vital roles in the Purkinje cell facilitation of survival in the juvenile quail (20) by suggested action via GABA-A receptor (21). Moreover, 3α-hydroxysteroid oxidoreductase (3α-HSOR) and 5α-reductase enzymes reduce the precursor steroid-found peripherally in skin and liver-to produce androstanediol, ALLO, and THDOC (22). Other substances are also involved in the induction of the biosynthesis of NASs: retinoic acids and vitamin D3 (VD3) induce neurosteroid production in human glial cells in culture (23), VD3 via induction of CYP11A1 and HSD3B1 (steroidogenic genes), which is mediated by vitamin D receptor (24).
Some factors affect the biosynthesis of NASs. The mRNA expression of 5α-reductase type I (5α-RI) is markedly downregulated (~50%) by social isolation in neurons of the cortex, hippocampus, and basolateral nucleus of the amygdala. For instance, 65-75% and smaller (~35%) decrease of 5α-RI mRNA levels were observed in dentate gyrus granule cells and CA3 glutamatergic pyramidal neurons, and frontal cortex pyramidal neurons (layer V/VI glutamatergic), respectively. Therefore, the anxiety and aggressive behavior seen in mice, which is socially isolated, is due to decreased ALLO biosynthesis in glutamatergic neurons of basolateral nucleus of the amygdala and frontal cortex (25) (Figure 1).

MeCHANiSMS OF ACTiON OF NASs
The mechanism of action of NASs can be classified as either (a) classical intracellular binding-in which this effect is described as a relatively slow genomic effects of NASs, (b) effect on membrane receptors and ion channels-in which this effect is also described as a rapid non-genomic effects of NASs, or (c) due to their metabolic interconversion to traditional steroids in the brain whereas some steroids led to rapid membrane effects through interaction with certain neurotransmitter receptors (6,26). In summary, NSs predominantly interact with ion channels and neuronal membrane receptors-but not primarily through interaction with intracellular receptors-and thereby modulate brain excitability (27), preferably through direct modulation of ion channels that are ligand-gated, remarkably GABA-A receptors (28).

ReGULATiON OF NASs
In the supraoptic nucleus, oxytocin regulates neurosteroid modulation of GABA-A receptors after parturition, since the activity of protein kinase C (PKC) and sensitivity of GABA-A receptor to ALLO in the supraoptic nucleus are mainly determined by the magnitude of activation of oxytocin receptor. Besides this, the GABA-A receptors are ALLO-resistant in breastfeeding mothers due to the presence of high level of oxytocin (29). The presence of relatively high concentrations of endogenous phosphatase during late pregnancy enhances the sensitivity of ALLO to GABA-A receptors. On the other hand, sensitivity of ALLO is restored by these phosphatases stimulation-which are endogenous-or PKC inhibition when NSs are becoming insensitive to GABA-A receptors (29). Instead, effect of the endogenous phosphatases and PKC on dissimilar phosphorylation sites or varying proteins of GABA, which are found on postsynaptic sites, may be prominent (30).

Effect on Gamma-Aminobutyric Acid-A Receptors
Gamma-aminobutyric acid-A receptors (GABA-A receptors) are major targets for central nervous system (CNS) actions of NSs (34). The GABA-A receptor function can be regulated by the NSs negatively or positively, based on the chemical structure of the steroid molecule (27,35). Secondary to the activation of the inotropic GABA-A receptors by NASs, chloride ion influx and causes neuronal membrane hyperpolarization (36).
The GABA-A receptors have novel subunit dependence of NSs action (34). The neurosteroid THDOC favorably augment the receptor with σ subunit among the seven different classes of subunits (α1-6, β1-3, γ1-3, σ1-3, δ, ε, θ). High (micromolar) and low (nanomolar) levels of NSs cause direct activation of GABA-A receptors and allosteric augmentation of GABA-regulated currents, respectively (37). However, changing in the modulation of NSs and inhibition of intact GABA are observed in neurons found in thalamic relay of mice without delta subunit (34). Inhibition of PKC activity abolishes the effect of THDOC on GABA-A receptors through increased α-4 subunit phosphorylation and its accumulation on cell surface by accelerating the α-4 subunit insertion into cell membrane without altering their endocytosis (38). The increased effect of THDOC on the phosphorylation of α-4 subunit and its expression on the cell surface is counteracted by S443 mutation, which is the major site of phosphorylation of PKC in the α-4 subunit (39).
Neurosteroids increased expression of α4βγ2 GAB-A receptors at Cornu Ammo (CA1) pyramidal cell synapses. The diminution in decay time for GABAergic miniature inhibitory postsynaptic currents following short-term NSs exposure is mediated by this increased expression-containing α4-GAB-A receptors localized to synaptic sites (40).
During stress, the balance between inhibition and excitation may be maintained by NSs, in which GABA-A receptor regulation is influenced by the levels of and duration of exposure to NSs (41). The plasma level of THDOC is elevated by about three times in the presence of stress (42). From the NASs, ALLO, THDOC, and androstanediol are powerful modulators of the GABA-A receptor (through positive allosteric modulation) and bring behavioral effects at low concentrations (1,43,44). Allopregnanolone was fully effective in suppressing GnRH release, mediated by interaction with the GABA-A receptor. Moreover, ALLO suppressive action on GnRH release in vitro is completely offset by GABA-A antagonistic NASs, PREG-S (31).
Enhancement of NSs due to reduced sensitivity of GABAergic synaptic transmission in dentate granule cells lead to the blockage of seizure propagation into the hippocampus (45). In women, seizure exacerbation during the perimenstrual period is contributed by the loss of NSs sensitivity of synaptic inhibition. In addition, withdrawal and elevated concentrations of NSs-observed in the mid-cycle-leads to exacerbation of seizure and anticonvulsant action, respectively (46). In the spinal cord, obliteration of induction of activity-dependent reflex plasticity is mediated by modulation of GABA-A receptors dependent inhibition as a result of the activity of the progesterone and its metabolites (ALLO and THDOC) (47).
Neurosteroids are potent cytoprotectants when they interact with neuronal GABA-A receptor. The potency and efficacy of NSs is affected by their structure. Sulfated NSs (PREGS and DHEAS) fully efficacious (about 70%) than the non-sulfated (AP and 17α-OH-AP). This is further substantiated by the structural activity analysis that indicated the association of an increase in potency but decrease in efficacy of the cytoprotectants with the lack of the double bond between C-5 and C-6 in AP and 17α-OH-AP and/or the hydroxyl group in the α-position (48).
Dehydroepiandrosterone and its sulfated metabolite, DHEA-S (more potent and more efficacious than the parent compound), which interacted with the picrotoxin/TBPS (t-butylbicyclophosphorothionate) binding site in a competitive manner (49), decreased GABA-A receptor-mediated responses on serotonin (5-HT) neuronal firing regulation, and vice versa. Androsterone and its parent compound (DHEA) can affect anxiety, cognition, and mood through enhancement of the GABA-A mediated response (50). Inhibition of NMDA receptor and potentiation of GABA-A receptor function, which will add on the clinical profile of anesthetic NASs-is mediated by another neurosteroid compound: (3α, 5β)-20-oxo-pregnane-3-carboxylic acid (3α5βPC). Notwithstanding, a NAS with a better clinical activity can be produced by augmenting blockage of NMDA receptor and reducing GABA-A receptor blockage since a direct correlation of the optimal property of anesthetic, anticonvulsant, and neuroprotective with high micromolar concentrations of 3α5βPC-needed for blocking effect on GABA-A receptors-might not be observed (51).
Stereospecific non-genomic activity on GABA-A receptor results in sharp increases in blood concentrations of 3α-OH-DHP-the neuroactive metabolite of progesterone-and ultimately leads to enhancing the baroreflex inhibition of brainstem rostral ventrolateral medulla neurons (52).

Effect on Gamma-Aminobutyric Acid-C Receptors
The interaction of NASs with the ρ1-GABA-C receptor-which is very specific chiral sites-and 3α configuration of GABA-C receptor is required for all steroid actions. NSs have multiple sites for interaction with the ρ1 receptor of GABA-C than GABA-A. However, a similarity in qualitative measurements and GABA-A receptor potentiation was observed in sites mediating 5β-reduced steroids inhibition and potentiation of the ρ1 receptor by 5α receptors (53).
Allopregnanolone, alphaxalone, and 5α-THDOC prolong the decay time and potentiate the GABA-induced currents. On the contrary, the ρ1-GABA-evoked current is inhibited by the co-administration of GABA with 5β-THDOC, pregnenolone, or 5β-DHP. The degree of inhibition and potentiation of ρ1-GABA provoked currents by NASs is reliant on the concentration of GABA. Since the application of GABA alone, following treatment with NAS, did not revert back to the control level for an extended period of time, a prolonged and persistent effect was observed on the effects of the NASs on ρ1 receptor channels. The 5α derivatives were potentiators (only at exceedingly low concentrations of GABA), whereas the 5β compounds were inhibitors of the GABA-evoked currents (54).

effect of NSs on N-Methyl-d-Aspartate Receptors
Neurosteroids (PS and PHS) control the NMDA receptors dependent (4) and independent (L-type calcium channeldependent) long-term potentiation (LTP) positively at a lower dose (1-5 mM) and negatively at a higher concentration (15 mM) (sigma-receptor function blockade) (55). Positive modulation is through increased Ca 2+ influx into presynaptic NMDA receptors (containing NR2D subunits) that raise the probability of glutamate discharge in hippocampal slices as studied in rats less than 6 days age (56,57). The positive regulation was observed to be negatively affected by antepartum ethyl alcohol exposure due to a change in NMDA receptor phosphorylation (58).
Pregnenolone sulfate stimulates a continued increase in the NMDA effect from 200 to 400%, by integration of extra subunit specific (σ1) receptors into the surface membrane (57,59). This integration requires G-protein-coupled activation of PKC and PLC and increased Ca 2+ ion (Figure 2) (59). The movement of NMDA receptors between the membrane and intracellular pools is required for maintenance and plasticity of synaptic connections; however, deregulation of the receptor movement has been associated with neuropsychiatric disorders (60). Hence, NMDA receptor surface expression plays a great role in disorders of NMDA receptor trafficking or provides a basis for the development of therapeutic interventions (59).

effect of NSs on voltage-Gated Calcium Channel Receptors
Effect on L-Type Voltage-Gated Calcium Channels Allopregnanolone (APα) (3α-hydroxy-5α-pregnan-20-one) promotes proliferation of hippocampal neural progenitor cells in rat and cerebral cortical neural stem cells (NSCs) in human at a nanomolar levels, and inhibits the proliferation of polysialylated form of the neural cell adhesion molecule (PSA-NCAM) at micromolar levels (62). This mechanism requires activation of L-type voltage-gated Ca 2+ channels (VGLCCs) (63). In mature neurons, APα increases chloride influx via allosteric modulation of the GABA-A receptor, thereby hyperpolarizing the neuronal membrane potential and decreasing neuron excitability (36). In marked contrast, it leads to an efflux of chloride in immature neurons, which causes depolarization of the membrane and opening of VGLCCs, then rises in intracellular Ca 2+ (63,64). Finally, this can trigger calcium-dependent mechanisms of mitosis in early precursor cells and human NSCs to promote neurogenesis (65).

Effect on T-Type Voltage-Gated Calcium Channels
The 5β-reduced NASs are powerful inhibitors of the T-type Ca 2+ channels in rat peripheral sensory neurons in vitro and very effective peripheral anti-pain agents in vivo, strongly proposing that T-type Ca 2+ channels are involved in peripheral somatic nociception (66). Thus, the 5β-reduced steroids are hopeful new agents for studying the role of T-type Ca 2+ channels in peripheral nociception and are potentially useful targets for the development of new pain therapies (67).

effect of NSs on Serotonin Receptor
Neurosteroids (DHEA) interact with ligand-gated serotonin (5-HT) receptors to enhance their firing action through σ1 receptors (68). This stimulates neurogenesis in the hippocampal dentate gyrus and shields it from glucocorticoids' detrimental attack (69).
Since DHEAs interact with σ1 receptors to bring their effect, σ1 receptor antagonists could eliminate the effect of DHEAs, whereas agonists of the receptor could simulate the blocking effect of DHEAs on 5-HT-evoked glutamate release via activation of Gi protein pyramidal cells of rat prelimbic cortex. At a lower concentration (1 µM), DHEAs could significantly hinder the 5-HT-evoked glutamate release in brain region by preventing its binding with 5-HT3. In reverse, DHEAs effect can be lessened with the rise in concentration due to promoting spontaneous glutamate release (70).
There is a relationship between central serotonergic activity and circulating ALLO concentrations. This is evidenced by an increase in ALLO concentrations in luteal phase after the administration of l-tryptophan in both controls and women with premenstrual syndrome (PMS) (greater increase in ALLO concentrations with PMS) (71).

effect of NSs on α 2 -Adrenergic Receptors
Neurosteroids (PREGS) blocks LTP of excitatory synapses in rat medial prefrontal cortex (mPFC) via interaction with α2adrenoreceptors secondary to enhancement of Gi proteins. After treatment of mPFC slices with the α2-adrenoreceptor inhibitor "yohimbine, " the blocking effect of PREGS on the induction of LTP was completely inhibited (72).

effect of NSs on Transient Receptor Potential Channels
Serotonin AP, DHEA Neurogenesis in hippocampal dentate gyrus (+) α2-adrenergic PS Blocks long-term potentiation (−) and epipregnanolone sulfate were found to potentiate TRPM3 activity. As compared to pregnenolone, PREGS shows comparable potency but greater than 10-fold higher intrinsic activity (74,75), and it stimulates TRPM3 activity via heat-dependent modulation (76).

effect of NSs on Reward Pathway via the σ1 Receptor
The σ1 receptor is an intracellular neuronal protein found in endoplasmic reticular, plasma, nuclear, and mitochondrial membranes. The ligands of σ1 receptor potently modulate intracellular Ca 2+ mobilizations and extracellular Ca 2+ influx (77,78). Pregnenolone, DHEA, and progesterone interfere with cocaineinduced reward path way in mice. DHEA and its precursor PREG facilitate cocaine-induced conditional place preference acting as σ1 receptor agonists (79).

effect of NSs on Liver X Receptors (LXRs)
Liver X receptors have two isoforms, LXRα (NR1H3) and LXRβ (NR1H2), classified under nuclear receptor superfamily. LXRα is highly found in liver and minimally in the intestine, macrophages, adipose tissue, lung, kidney, and adrenal gland; whereas LXRβ is broadly expressed (80). Oxysterols (oxidized cholesterol) bind with LXRs and induce expression of genes which eradicate harmful cholesterol level by efflux through ATP-binding cassette family of transporters (81). Activation of LXRs promote cholesterol disposal, steroidogenesis in the adrenal gland, regulation of StAR expression, restores normal StAR mRNA levels and completely restores the mRNA levels of P450scc to non-diabetic levels, and raises the local levels of NASs like PROG and DHP. These functions play a role in protecting diabetes patients from peripheral neuropathy (82).

effect of NSs on voltage-Dependent Anion Channels (vDAC)
Interactions of NASs with VDAC isoforms (prominent brain protein) are important to regulate mitochondrial function, synaptic plasticity, or apoptosis. A role in avoiding apoptosis explains the neuroprotective actions of the NASs (83).

effect of NSs on Microtubule-Associated Protein 2 (MAP2)
Pregnenolone and its synthetic analog-MePREG interacts with MAP2 at an unknown binding site. Then, it stimulates microtubule polymerization (rat brain and from PC12 cells) and the extension of neuritis in pheochromocytoma cells that are exposed to nerve growth factor (NGF) (84).

effect of NSs on Neurotrophin NGF Receptors
Acting as a neurotrophic factor, DHEA affects neuronal survival and neurogenesis during development and in aging in the brain via interacting with pro-survival TrkA and pro-death p75 (NTR) membrane receptors of neurotrophin NGF. This stimulation inhibits the apoptotic loss of NGF receptor positive sensory and sympathetic neurons. Furthermore, it provides a mechanistic explanation for the multiple actions of DHEA in other peripheral biological systems expressing NGF receptors, such as the immune, reproductive, and cardiovascular systems (85) ( Table 1).

iNvOLveMeNT OF NASs iN NeUROLOGiCAL DiSeASeS
Epilepsy-related with menstruation (catamenial epilepsy) currently has no specific approved treatments. This kind of epilepsy is as a result of enhanced excitability due to withdrawal of NSs, which in turn leads to upregulation of α4 subunit and linked with upregulation of Egr3 and reduced synaptic inhibition. In addition, neurosteroid withdrawn mice were amazingly less sensitive to the antiseizure effects of diazepam, and progesterone receptor (PR) knockout animals were also less sensitive to the protective actions of diazepam during neurosteroid withdrawal (86). Neurosteroid (ALLO) has broad-spectrum anticonvulsant activity and recently it is approved for investigational use to treat an individual with prolonged super refractory status epilepticus (87). Other NS like APα also has anxiolytic and sedative-hypnotic properties with no indicated toxicological adverse events in healthy human volunteers and in children with refractory infantile spasms (88)(89)(90). Further investigations are presently ongoing to identify the neurogenic potential of APα in rodent models of aging and Alzheimer's disease (AD) (63). The concentrations of testosterone, cortisol, PROG, DHEA, DHEAS, and estrogen levels have been found to be changed in some patients with schizophrenia. The level of DHEA contrariwise associated with negative symptom severity in drug-free men with first-episode psychosis (91). In major depressive episodes, NS, mainly ALLO and pregnenolone, are found to be diminished in both the cerebrospinal fluid (CSF) and the plasma of untreated patients (25). However, following effective psychopharmacological treatment concentrations of ALLO in depressed patients increase to normal levels. Several findings support the hypothesis of an antidepressant effect of ALLO (92).
Stress-induced groups of rats showed declined amount of DHEA-S, which indicates that DHEA may play an important role in the development of adaptive responses to a stressful event (93). Higher blood level of DHEA and DHEAS were related with less experience of symptoms in attention deficit hyperactivity disorder (ADHD), in particular, hyperactivity symptomatology. However, the effect in ADHD patients remains elusive (94). Alterations in gene expression of the enzymes which synthesize NSs may be involved in the pathology of AD. In early AD, there is an attempt to increase the biosynthesis of NSs and NASs through increased mitochondrial import of cholesterol (95).
The amount of AP and DHEA showed significant reduction in specimens of multiple sclerosis white matter compared to controls. Allopregnanolone has a role in controlling neuroinflammation and protection of demyelination and axonal loss in models of multiple sclerosis by interacting with GABA-A receptor. Experimental autoimmune encephalitis (EAE) is characterized by disordered neurosteroidogenic machinery, which causes reduced expression of the enzymes (3-alpha-hydroxysteroid dehydrogenase) involved in AP biosynthesis, related with reduced AP levels in the CNS. Since AP has a role in controlling neuroinflammation and protection of demyelination and axonal loss, it also used for the treatment of EAE associated myelin and axonal injury (96).
Levels of allopregnanolone and 5-DHP were found reduced in the CSF of Parkinson's disease (PD) patients signifying a part for these progesterone metabolites in the disease. Moreover, the enzymes which synthesize allopregnanolone, mRNA expression of 5-reductase type 1 (SRD5A1) was significantly reduced in peripheral blood mononuclear cells of PD patients, proposing a generalized defect in the enzymatic machinery that regulates the metabolism of progesterone (97).

AUTHOR CONTRiBUTiONS
KT and TA conducted the review and edited the manuscript.

FUNDiNG
The study was not supported by any funding agent.