Allopregnanolone and its antagonist modulate neuroinflammation and neurological impairment

several brain disorders, either as a secondary consequence or as a primary


Introduction
Inflammation has traditionally been viewed as a secondary consequence of disease, against which therapeutic interventions are usually directed.However, inflammation, in particular neuroinflammation, is increasingly recognized as an important driver of disease-related morbidity and a legitimate therapeutic target.
It is well known that steroids, especially glucocorticoid steroids, are used as treatment for and modulators of inflammatory disorders.What is less well appreciated are the roles in neuroinflammation of Positive Allosteric Modulating steroids (steroid-PAM) of the Gamma-Aminobutyric Acid-A (GABA-A) receptor such as allopregnanolone (ALLO, and their antagonist GABA-A modulating steroid antagonists (GAMSA).Perhaps also not well known are the roles of GABA and GABA-A receptors in regulating inflammation and inflammatory cells, particularly in the brain.This review therefore focuses on the complex role of steroid-PAMs and GAMSA, in neuroinflammation as well as the rationale for modulating the pleiotropic effects of neurosteroids such as ALLO as a potential therapeutic strategy against neuroinflammation.
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Neuroscience and Biobehavioral Reviews
In this context, it is also important to point out what this review does not cover.For example, GABA-A receptor modulators have multiple effects in the brain, only some of which depend on their effects on neuroinflammation.For information regarding the effects of GABA-A receptor modulators unrelated to neuroinflammation, we refer readers to other reviews (Diviccaro et al., 2022, Guennoun et al., 2015).Similarly, a comprehensive summary of all steroid groups which modulate inflammation is beyond the scope of this review.For information pertaining to other steroid groups, we would therefore direct the reader to papers pertaining to adrenal hormones and their role in neuroinflammation (Hassamal, 2023) as well as papers pertaining to glucoand mineralo-corticoid steroids, androgen and estrogen steroids (e.g.; Vegeto et al., 2008;Do Rego et al., 2012;Silverman and Sternberg, 2012;Ericson-Neilsen, and Kaye, 2014;Cain and Cidlowski, 2017;de Kloet et al., 2018;De Nicola et al., 2018;Vandewalle, et al., 2018;Yilmaz et al., 2019;Hassamal, 2023), including those which describe the effects of glucocorticoids on proinflammatory signaling pathways and resolution of the inflammatory response through programming effects on inflammatory cells (Cain and Cidlowski, 2017).
The GABA-system is known to be involved in regulation of immune responses and most types of immune cells express GABA-A receptors (i.e., monocytes and macrophages), making them potential targets for GABA-A receptor active steroid-PAM and GAMSA.(Bhandage et al., 2015Reyes-Garcia MG, 2007, Stuckey et al., 2005, Backstrom et al., 2011Llansola et al., 2024).GABA has been shown, for example, to influence immune cell proliferation, migration, and cytokine secretion (Jin et al., 2013, Bhat et al., 2010) and to inhibit the proliferation of T-cells, inhibit immune responses and decrease inflammatory cytokine production in peripheral macrophages (Reyes-Garcia MG, 2007) through functional GABA-A receptors (Bhandage et al., 2018, Tian et al., 2011, Prud'homme et al., 2015).Given these effects of the GABAergic system on inflammatory responses (Wu et al., 2017), it is of interest to investigate whether GABA-A receptor modulators, steroid-PAMs and GAMSA influence the immune response.In this review, we focus on elucidating the involvement of GABA's, steroid-PAM's and GAMSA's in neuroinflammation.
We are aware of comparatively few studies on the role of steroid-PAMs in neuroinflammation.We have therefore concentrated our review on different animal models of liver injury and hepatic encephalopathy (HE), including porta-caval shunt (PCS), NH 4 Cl administration and bile duct ligation (BDL), all of which result in neuroinflammation and encephalopathy and serve as a model of how systemic disorders may result in neuroinflammation, as well as studies in healthy adults and patients with cirrhosis.The studies we cite include some of our own.One additional reason for emphasizing HE models is that ALLO and pregnanolone levels are increased in these animal models in the periphery as well in the brain, which is like what has been reported in humans (Cabrera-Pastor et al., 2019, Ahboucha et al., 2006, Butterworth et al., 2009, Cauli et al., 2009).Indeed, the effects of steroid-PAMs on the GABA-A receptor are especially interesting as there is evidence that GABA and GABA-A receptors are important for modulation of inflammatory actions in the periphery as well as in the brain.Information pertaining to this, and other issues, are discussed below and organized by topic.

Neuroinflammation mechanisms and role in neurological disorders
Neuroinflammation is a complex inflammatory process in the central nervous system in which the brain's innate immune system is activated in response to a pathogenic or traumatic challenge.This challenge may be induced by a variety of causes from within or outside the central nervous system, including sustained peripheral inflammation, infections, traumatic or ischemic brain injury or exposure to toxic agents (Ransohoff et al., 2015).
A key step in the neuroinflammatory process is activation of microglia (Carson et al., 2006a,b).Microglia are the brain's permanent macrophages and play a crucial role in the brain's defense and repair as well as in neuroinflammation in many neurological and neurodegenerative diseases (Harry and Kraft, 2012).Microglia become activated in response to alterations in neuronal function or neuronal damage or in the presence of pathogens, infiltration into the brain of cells from the peripheral immune system, neurotoxins, infection, or brain injury (Schetters et al., 2018;Glass et al., 2010;Ransohoff and Khoury, 2015).When activated, microglia change their morphology, proliferate and secrete pro-inflammatory cytokines, such as TNFα, IL-1β, IL-6, and IL-23, chemokines, reactive oxygen species (ROS) and nitric oxide (NO) and express MHCII (Saijo and Glass, 2011;Glass et al., 2010;Ransohoff and Khoury, 2015).Astrocytes, which normally have a major neuro-supportive, homeostatic role in the healthy brain, adopt a more reactive state during acute or chronic adverse conditions.Under pathological conditions, astrocytes also become activated, undergoing morphological and molecular changes to adopt so-called "reactive" states, which alter their function and enhance their neurotoxic properties at the expense of reduced neuro-supportive properties and homeostatic functions (Patani et al., 2023;Escartin et al., 2021;Lattke and Wirth, 2017;Sofroniew, 2020;Sofroniew and Vinters, 2010).Activated astrocytes are widespread and typically found in the brain of patients who died with Alzheimer's, Parkinson`s Disease (PD) or as a result of advanced liver disease.Once activated, astrocytes also produce pro-inflammatory signaling molecules, including cytokines, growth factors, and complement molecules (Patani et al., 2023;Balzano et al., 2018;Leone et al., 2023) The cytokines released by activated microglia or astrocytes, such as IL-1β, IL-6, and TNFɑ, are the most important neuroinflammatory signaling molecules and affect neurophysiological mechanisms modulating cognition and memory (Patani et al., 2023;Hong et al., 2016).For example, rats with chronic hyperammonemia show neuroinflammation in the hippocampus, with microglia and astrocytes activation and increased IL-1β levels.Enhanced activation of IL-1 receptors in hippocampal neurons by increased IL-1β levels triggers the activation of signal transduction pathways that result in altered membrane expression of the AMPA and NMDA glutamate receptors, leading to cognitive impairment.Reducing the activation of the IL-1 receptor with IL-1R antagonist reverses these changes and restores cognitive function (Taoro-Gonzalez et al., 2018, 2019).Increased levels of TNFα activates other signal transduction pathways, leading to impairment of spatial reference memory (Cabrera-Pastor et al., 2016).
Repeated restraint stress also induces neuroinflammation and increases TNFα levels in rats, which contribute to stress-induced cognitive impairment (Azadbakht et al., 2015).Chronic stress leads to neuroinflammation, with activation of microglia, release of pro-inflammatory molecules, and the recruitment of peripheral immune cells to the brain, creating the inflammatory environment characteristic of many neurological disorders.Stress is associated with mood changes and mild cognitive impairment, leading to an increased risk of developing psychiatric and neurological disorders (Harry and Kraft 2008;Meyer, 2014).

GABA and GABA-A receptor (GABA-AR) involvement in inflammation
Multiple neurotransmitters, including gamma-Aminobutyric acid (GABA), have been shown to modulate the immune system (Prud 'homme et al., 2015).Increasing evidence points to neuroinflammations effects via the GABAergic system as an etiological factor for neurological and psychiatric illness (Bannister, 2019).The expression of GABAergic components has been observed in several immune cell types, including microglia and activated astrocytes, T-lymphocytes, myeloid lineage cells, and NK cells, (Wu et al., 2017).In most cases GABAergic signaling has been reported to have anti-inflammatory and immunosuppressive effects by directly suppressing the functions of certain peripheral immune cells, such as macrophages and lymphocytes (Bhandage et al., 2018, Kim et al., 2018).Accumulating evidence suggests that GABAergic neuron number is altered under inflammatory conditions (Falco et al., 2014;Rossi et al., 2011Rossi et al., , 2012) ) and most of these studies suggest that inflammation decreases GABAergic neuron density (Crowley et al., 2016).On the other hand, the proinflammatory factor TNF-α increased the synaptic and total GABA-A receptor number in the membrane of motor neurons suggesting an increase in GABAergic capacity by the proinflammatory factor.However, the effect of TNF-α on GABA-AR trafficking was complex, displaying a nonlinear dose-dependent relationship (Stuck, et al., 2012).
These complex effects of inflammation need to be considered in the context of GABA-A receptor function.GABA-A receptors are chloride channels comprising five subunits belonging to 19 different isoforms known as α1-6, β1-3, γ1-3, δ, ε, θ, π, and ρ1-3 (Olsen and Sieghart, 2009).In adults where intracellular concentration is low, chloride influx through GABA-AR inhibits the firing of neuronal action potentials and underlies GABA's sedative effects.In contrast, during fetal development the intracellular Cl-concentration is comparably high, and because of that activation of GABA-A-receptors causes excitation (Kahle et al., 2008).However, elevated intracellular Cl-concentrations can also produce GABA-evoked excitability in the adult brain, both in postsynaptic (Khirug et al., 2008;Szabadics et al., 2006;Martina et al., 2001;Gulácsi et al., 2003;Moenter and DeFazio, 2005) and in presynaptic terminals (Haage et al., 2002).The intracellular Cl-concentration is determined by the activity of inward and outward directed transmembrane Cl-pumps, where the major inward pump is NKCC1 and the major outward pump is KCC2 (De Koninck, 2007;Price et al., 2005Price et al., , 2009)).In adult animals and probably also in humans, the outward directed pump KCC2 dominates, keeping the intracellular Cl-concentration low.However, inflammation affects the expression of chloride transporters and influences directionality of GABA signaling, and those inflammatory cytokines, in particular interleukin 1β (IL1), may represent key causal factors for this phenomenon (Pozzi et al., 2020).Proinflammatory cytokines have been demonstrated in the pathological contexts where reduction of KCC2 has been found to be involved e.g. the pro-inflammatory IL-1β is recognized to contribute to epilepsy development (Vezzani and Baram, 2007), and the therapeutic effect of a IL-1β antagonist, is effective in febrile infection-related epilepsy syndrome (Dilena et al., 2019), It is known that activated microglia, increase NF-κB activation and pro-inflammatory cytokines such as TNFα, IL-6, and IL-1β (Fuzzati-Armentero et al., 2019;Ji et al., 2022;Tansey et al., 2007;Caggiu et al., 2019).In neuropathic pain, for example, the Cl-pump KCC2 is less efficient (Price et al., 2005).That the activity of chloride pumps can change in adulthood is also shown in human brain tissue close to epileptic foci in patients (Huberfeld et al., 2007;Munoz et al., 2007).The results suggest that under certain situations and in certain conditions, GABA is excitatory.
The change in direction of GABAergic signaling may be the reason for unexpected paradoxical anxiogenic response to GABA stimulation seen especially at low doses/concentrations of positive GABA-A receptor modulators (Backstrom et al., 2011).An example is women with premenstrual dysphoric disorder (PMDD) whose GABA-A receptor sensitivity are altered, and they manifest an unexpected GABAergic response to allosteric modulators compared with healthy subjects (Backstrom et al., 2014).Similar response to steroid-PAM is observed in female pubertal rats (Shen et al., 2007)

Enhanced GABAergic neurotransmission may modulate neuroinflammation and associated impairment in some neurological diseases
The induction of cognitive and motor impairment by neuroinflammation is mediated by alterations in neurotransmission.For example, as described above, increased IL-1β levels in the hippocampus alter glutamatergic neurotransmission in the hippocampus leading to cognitive impairment in hyperammonemia (Taoro-Gonzalez et al., 2018, 2019).Neuroinflammation-induced alterations in glutamatergic neurotransmission are also involved in other pathologies including Alzheimer`s and Parkinson´s disease (Czapski and Strosznajder 2021;Iovino et al., 2020).
Alterations in GABAergic neurotransmission also contribute to the cognitive and motor alterations in neuroinflammation-associated pathologies.Neuroinflammation may increase GABAergic neurotransmission and, to add to the complexity, when immune cells possessing GABA-A receptors are stimulated with GABA, they change the strength of the CNS inflammation (Tian and Kaufman, 2023).For example, in rats with hyperammonemia-induced hepatic encephalopathy, neuroinflammation increases GABAergic neurotransmission by activating the TNFα-TNFR1-NF-kB-glutaminase-GAT3 and the TNFα-TNFR1-S1PR2-CCl2-BDNF-TrkB pathways.This is associated with induction of motor incoordination, which is reversed when GABAergic neurotransmission is normalized by blocking these pathways (Cabrera-Pastor et al., 2018;Arenas et al., 2022aArenas et al., , 2022b) ) There is an interplay between GABAergic neurotransmission and neuroinflammation, which modulate each other leading to the cognitive and motor impairment in some pathologies, including Parkinson's disease, multiple sclerosis and hyperammonemia and hepatic encephalopathy (Paul et al., 2014;Crowley et al., 2016;Agusti et al., 2016Agusti et al., , 2017;;Heo et al., 2020;Dadsetan et al., 2016b).The interplay also has implications for fatigue and mood disorders (D'Mello and Swain 2014).
Both pro-inflammatory and anti-inflammatory effects have been proposed for GABA.Heo et al. (2020) showed that in animal models of Parkinson's disease the levels of GABA are increased in substantia nigra pars compacta, especially in activated astrocytes.These increased levels of GABA enhance the activation of GABA-A receptors leading to reduced expression of tyrosine hydroxylase in neurons that, in turn, is responsible for the motor deficits in these animal models.This effect occurs in neurons that have not yet died, and it is therefore possible to reverse these mechanisms to improve motor deficits and quality of life of the patients.Heo et al. (2020) have also shown that inhibiting MAOb reduces GABA levels and restores the expression of tyrosine hydroxylase in neurons, which is associated with improvement of motor deficits.Tyrosine hydroxylase expression is also restored by blocking GABA-A receptors containing the α5 subunit, supporting the hypothesis that enhanced activation of GABA-A receptors mediates the inhibition of tyrosine hydroxylase expression and the motor deficits.Lang et al. (2020) proposed that GABA activates microglia and that GABA-mediated activated microglia induce neuroinflammation in the hippocampus of mice following cold exposure through the NLRP3 inflammasome and NF-κB signaling pathways.
Enhanced GABAergic neurotransmission also contributes to induction of neuroinflammation in hyperammonemia and hepatic encephalopathy.Treating hyperammonemic rats with bicuculline, an antagonist of GABA-A receptors, reduces neuroinflammation in hippocampus and cerebellum and improves cognitive and motor function (Malaguarnera et al., 2019(Malaguarnera et al., , 2021)).This further supports the view that excessive GABAergic neurotransmission may induce neuroinflammation and associated impairment of cognitive and motor function in different pathological situations.

Allopregnanolone modulates GABAergic neurotransmission and neuroinflammation
In addition to the direct activation of GABA-A receptors by extracellular GABA, activation of these receptors is modulated by other mechanisms including steroid-PAMs and changes in GABA-A receptor membrane expression in response to neuroinflammation (Arenas et al., 2022).
Steroid-PAMs comprise a family of allosteric GABA-A receptor modulators.Some steroid-PAMs such as allopregnanolone (ALLO) or tetrahydrodeoxy-corticosterone (THDOC) enhance, while others such as pregnenolone-sulfate reduce GABA-A receptor activation.Gonzalez-Usano et al., (2014) showed that pregnenolone-sulfate reduces GABAergic neurotransmission and restores cognitive function and motor coordination in hyperammonemic rats.
The effects of steroid-PAMs differ depending on GABA-A receptor subunit composition.For example, ALLO enhances activation of most GABA-A receptors but the effect on extra-synaptic, α4βxδ, receptor subtypes is much stronger than on intra-synaptic, e.g., α1, GABA-A receptor subtypes (Belelli and Lambert, 2005).
Steroid-PAMs may therefore enhance or reduce GABAergic neurotransmission, just as they may also enhance or reduce neuroinflammation.Potentiation of GABA-A receptors activation by ALLO may contribute to induction of neuroinflammation, while ALLO may also reduce neuroinflammation by other mechanisms (see below).

TSPO is a marker of neuroinflammation
One of the most frequently used biomarkers of neuroinflammation in vivo is imaging of Translocator Protein 18 kDa (TSPO, previously known as "peripheral type benzodiazepine receptor" PTBR) (Corica et al., 2023).TSPO expression is increased in activated microglia and astrocytes, making TSPO a good biomarker of glial activation and neuroinflammation independent of the etiology (Chen and Guilarte, 2008;Venneti et al., 2006;Rupprecht et al., 2010;Liu et al., 2015).TSPO is also involved in steroid-PAM synthesis in the brain (see below).Many neurological and neurodegenerative diseases and psychiatric disorders (Alzheimer`s disease, hepatic encephalopathy) show increased expression of TSPO especially in activated microglia (Cagnin et al., 2001a;Werry et al., 2019;Knezevic, Mizrahi, 2018;Lavisse et al., 2012).

TSPO modulates synthesis of steroid-PAMs in the brain
TSPO is a high-affinity cholesterol-binding protein (Midzak et al., 2015;Li, 2016).Cholesterol is a precursor molecule for the synthesis of steroid compounds in the mitochondria of steroidogenic cells.TSPO mediates the transport of cholesterol across the mitochondrial membrane and thus promotes the local production of steroid compounds (Midzak et al., 2016).The conversion of cholesterol to pregnenolone in the mitochondrial matrix through cytochrome P450 (CYP11 A1) activity is the first step in the synthesis of various steroid compounds like progesterone (Black et al., 1994;Farkash et al., 1986;Fan et al., 2015;Agis-Balboa et al., 2006).Locally synthesized or peripherally derived progesterone is thereafter metabolized to dehydroprogesterone (DHP) and ALLO.The steroidogenic acute regulatory protein (StAR), together with TSPO, is a rate-limiting step in the transfer of cholesterol to mitochondria and production of steroid-PAMs (Rone et al., 2009;Krueger and Papadopoulos, 1990;Black et al., 1994;Farkash et al., 1986).Steroid-PAM production is related to the concentration of TSPO (Papadopoulos et al., 2015(Papadopoulos et al., , 2018)).That is, the local brain concentration of steroid-PAMs can become very high when TSPO expression is increased, as with neuroinflammation.However, steroid-PAM production is not totally dependent on TSPO.Studies in TSPO knockout mice show that cholesterol can be transported into mitochondria without TSPO.(Morohaku et al., 2014;2018).Up-regulation of StAR and other transport-related proteins partly compensates for the absence of TSPO, but cholesterol transport is greatly reduced, and steroid-PAM synthesis was reduced (Germelli et al., 2021;Stocco et al., 2017).There are also other ways apart from TSPO-regulated synthesis that steroid-PAM synthesis and brain concentrations can be influenced and regulated in the brain, including other neurotransmitters, neuromodulators and neuropeptides (for more information c.f. Giatti et al., 2019, Porcu et al., 2016, Do Rego et al., 2012, Raux and Vallee, 2023).We emphasize TSPO in this review because it is closely linked to neuroinflammation and activated microglia.Steroids produced in the periphery by endocrine organs pass easily through the blood brain barrier, and their concentration in brain therefore reflects to a certain degree their concentration in the periphery.However, the reverse is not necessarily true.That is, neurosteroids produced locally in the brain may be present in high concentrations in the area where they were produced, but changes in localized brain neurosteroid concentrations may not be reflected in peripheral blood.

Allopregnanolone levels in brain in neuroinflammationassociated pathologies
The production of steroid-PAMs may increase during neuroinflammation because of TSPO's increased activity and steroid-PAMs reach their maximal levels of site-specific de novo synthesis in the brain areas showing activated microglia and neuroinflammation (Germelli, 2021).An upregulation of TSPO increases the binding and transport capacity of cholesterol into the mitochondria and steroid-PAM synthesis (Lejri et al., 2019;Stocco et al., 2017;Black et al., 1994).An increase of TSPO levels occurs, for instance, with increased neuronal activity (Notter et al., 2021).Treatment with TSPO ligands increases the synthesis of steroid-PAMs such as pregnenolone and ALLO (Liere et al., 2017;Rupprecht et al., 2009;Primofiore et al., 2004).TSPO stimulation also increases the steroid-PAM-mediated release of Brain-Derived Neurotrophic Factor (BDNF) and induces an increased inhibitory GABAergic tone in neuroinflammatory areas in the brain.(Germelli, 2021).
Another factor of interest is the inflammation in the gut.There is evidence that the microbiome in the gut communicates with the brain and influences emotional behavior and stress responses.Steroid-PAMs such as ALLO seem to be involved in the gut-brain signaling process; in particular ALLO, which is produced in the gut as well as in brain and may traffic between the gut and the brain (Pinna, 2023).
Upregulation of TSPO may lead to increased production of steroid-PAMs in the brain in pathologies associated with neuroinflammation (Table 1).In summary, these findings collectively suggest that in some disorders affecting the brain, biosynthesis of neurosteroids is increased via increased mitochondrial cholesterol input in some regions of the brain, and that this increase may contribute to neuroinflammation, for example in AD (Luchetti et al., 2011).
CNS steroid-PAM levels as well as the production and effect of steroid PAMs may also differ depending on the etiology of the neuroinflammation.One hypothesis holds that neuroinflammation originating in the brain results in early neuronal inflammation (Table 1, Luchetti et al., 2011, Luchetti et al., 2023) and, further, that when the CNS cells affected by the inflammation are destroyed, the production of and effects of steroid-PAMs are eliminated or reduced.By contrast, when neuroinflammation is the result of inflammation originating in the periphery, the destruction of cells in the CNS is delayed and, consequently, the production and effects of steroid-PAMs are comparatively delayed and prolonged.In certain liver advanced disorders, for example, the metabolism of steroids is changed leading to increases in peripheral and, subsequently, CNS concentrations (Table 1, Ahboucha andButterworth, 2008, Wetten et al., 2022).This hypothesis will be further discussed below, using HE as example of a peripheral steroid-PAM source and PD and MS as intra CNS production as source of steroid-PAM.
In a model of peripheral origination disorder, rats with portacaval shunts (PCS), a model of hepatic encephalopathy (HE) with increased peripheral and brain steroid-PAM concentrations (Ahboucha and Butterworth, 2008), show neuroinflammation (Agusti et al., 2011) as well as increased TSPO expression (Agusti et al., 2014).ALLO also increases in the brain in another model of HE; i.e., rats with chronic hyperammonemia (Cauli et al., 2009).Similarly, in humans, Cagnin et al. (2006) showed that cirrhotic patients with liver failure complicated by hepatic encephalopathy (HE) show increased binding of ligands of TSPO.The levels of ALLO are also increased in human postmortem cerebral cortex of cirrhotic patients who died with HE compared to controls without HE (Ahboucha et al., 2006).Similar increases in both ALLO and another positive modulator of GABA-A receptors, namely THDOC, have been reported in other models of HE, including acute liver failure with HE (Ahboucha et al., 2012;Norenberg et al., 1997).Thus, increased production of the steroid-PAM ALLO may contribute to some of the cognitive manifestations of HE (Johansson et al., 2015;Montagnese et al., 2021).
Blood levels of ALLO are also increased in patients with another liver disorder, Primary Biliary cholangitis/cirrhosis (PBC, previously called Primary Biliary Cirrhosis), suggesting that the brain levels are even higher (Ahboucha and Butterworth, 2008;Wetten et al., 2022).PBC is an autoimmune disease with a female predominance characterized by inflammation of bile ducts, and, in some patients, development of fibrosis and cirrhosis (Hirschfield and Gershwin, 2013).The symptoms of PBC include fatigue, itchy skin, dry eyes and mouth, abdominal pain.Some patients also experience personality changes and impaired cognitive function, memory, processing information and concentration that they describe as "brain fog" which is disproportionate to the severity of their liver injury and typically occurs prior to development of cirrhosis (Grover et al., 2016;Newton et al., 2008;Wunsch et al., 2021).Neuroinflammation is probably responsible for these neuropsychiatric symptoms, especially fatigue, in PBC (Lynch et al.;2022).There are no licensed or effective medications for the management of fatigue in PBC (Phaw et al.;2020).
Reduced levels of steroid-PAMs have also been reported in some pathologies associated with neuroinflammation (Table 1).Disruption of steroid-PAM synthesis and decreased levels of dehydroepiandrosterone and ALLO have been reported in brain in patients with multiple sclerosis (MS) as well as in an animal model of MS: experimental autoimmune encephalomyelitis (EAE) (Noorbakhsh et al.;2011;Boghozian et al., 2017;Leva et al., 2017).In the EAE model of MS, increasing the production of the steroid-PAM ALLO may contribute to neuroprotection and tissue repair in MS and/or may be involved in cognitive impairment.Treatment of mice and rat EAE models with the TSPO agonist, XBD-173, increased the levels of steroid-PAMs in the brain (Ravikumar et al., 2016).
Decreased progestin levels (e.g.ALLO) have been associated with late stages of Parkinson's disease (PD).For example, ALLO levels are reduced in the CSF of PD patients (di Michele et al., 2013).In the PD model of 6-OHDA-treated male rats, pregnenolone and dihydroprogesterone levels are reduced in the striatum and cortex, respectively (Melcangi et al., 2012).
In a recent study ALLO levels were measured in the substantia nigra (SN) and prefrontal cortex (PFC) in controls and patients at different stages of PD.Postmortem brains from PD patients with early disease (PD4, preclinical to initial symptoms) and advanced disease (PD6) were included (Braak et al., 2003).The concentration of ALLO was increased in early PD4 compared to controls, but 5α-DHP and ALLO levels were decreased in PD6 compared to PD4 (Luchetti et al., 2023).The metabolite of ALLO, 3α5α20α-hexahydroprogesterone (3α5α20α-HHP) was increased in PD4 compared to controls.In the PFC, 3α5α20α-HHP was higher in PD4 compared to both control and PD6.3α5α20α-HHP has a 3α-OH-5α structure and is thus most likely a positive allosteric modulator of the GABA-A receptor (Rahman et al., 2008).
These findings suggest that upregulation of ALLO synthesis occurs in the early stages of PD, followed by a downregulation at later stages (Luchetti et al., 2023).The concentrations and effects of steroid-PAMs are thus dependent on the stage of the disease.Similar findings have been reported for steroid-PAMs during the progression of Alzheimer's disease (Luchetti et al., 2011).
The presence of high steroid-PAM levels locally was recently reported in the 6-OHDA model of Parkinson`s disease in rats.Inhibiting 5α-reductase, the critical enzyme in steroid-PAMs synthesis, decreases ALLO levels and reduces L-DOPA-induced dyskinesia in these Parkinsonian rats (Frau et al., 2017;Fanni et al., 2019).Dyskinesia is a troublesome complication of chronic L-DOPA treatment with different abnormal involuntary movement symptoms, named L-DOPA-induced dyskinesias (LIDs).Nearly all patients with PD eventually develop LID (Ahlskog and Muenter, 2001;Turcano et al., 2018).Pregnenolone dose-dependently counteracts the development of LIDs in a rat model of PD without affecting the efficacy of L-DOPA.The data suggest that treatment with pregnenolone restores the imbalance in striatal D1R-related signaling (Corsi et al., 2023).Pregnenolone also acts as an anti-inflammatory and/or immunoregulatory agent in neuroinflammatory diseases (Murugan et al., 2019).

Effects of steroid-PAMs on neuroinflammation
Neuroinflammation may be modulated by steroid-PAMs.Peripheral steroids synthesized in the gonads or adrenals easily cross the bloodbrain barrier.However, endogenous steroid-PAMs synthesized locally may reach high site-specific concentrations, thereby predominating over peripheral steroids and exerting region-specific effects (Yilmaz et al., 2019).For example, in the PCS rats model of hepatic encephalopathy, TSPO increases differentially in different brain areas (Agusti et al., 2014).
As discussed above, enhanced GABAergic neurotransmission may induce neuroinflammation and associated neurological impairment in some pathologies.ALLO may therefore induce pro-inflammatory effects through potentiation of activation of GABA-A receptors.
However, ALLO may also reduce neuroinflammation by other mechanisms.For example, Keitel et al. (2010) showed that ALLO is also an agonist of Takeda G protein-coupled receptor 5 (TGR5), a membrane-bound bile acid receptor in the gastrointestinal tract and immune cells which is also expressed in neurons and glial cells.TGR5 mRNA is downregulated in cultured rat astrocytes in the presence of steroid-PAMs or ammonia as well as in cerebral cortex from cirrhotic patients who died with hepatic encephalopathy (HE).Keitel et al. (2010) conclude that TGR5 is a steroid-PAM receptor in the brain with implications for the pathogenesis of HE.In cultured microglia, ALLO reduces the activation and the increase in pro-inflammatory cytokines induced by lipopolysaccharide (LPS) through activation of TGR5, indicating that ALLO may reduce neuroinflammation through activation of TGR5 (Karababa et al., 2017).Activation of TGR5 by selective agonists also reduces neuroinflammation in an MPTP mice model of Pakinsons's disease (Huang et al., 2022), suggesting that ALLO may also reduce neuroinflammation in some pathological situations through TGR5 activation.
Multiple sclerosis (MS) is associated with both neuroinflammation and neurodegeneration (Sospedra and Martin, 2005;Trapp and Nave, 2008).MS is characterized by leukocyte infiltration into the brain followed by myelin damage, local gliosis, and axonal damage.Some reports suggest beneficial effects of steroid-PAMs in the treatment of MS.In the EAE mouse model of MS, progesterone treatment improved disease severity (Arnason and Richman, 1969a,b;Garay et al., 2007).Progesterone also stimulates myelin formation in the peripheral nervous system (Baulieu and Schumacher, 1997).Schwann cells have GABA-A receptors and respond to ALLO treatment by upregulating myelin proteins, i.e., myelin protein 22 and P0 (Melcangi et al., 1999) and perhaps oligodendrocyte myelin basic protein (Ghoumari et al., 2003).In the EAE animal model of MS, ALLO treatment reduced neuroinflammation and disease burden and limited the development of the pathology (Noorbakhsh et al., 2011;Daugherty et al., 2013;Nezhadi et al., 2016;Jolivel et al., 2021).
Neuroinflammation and associated 'reactive' microglia have long been recognized as elements of PD (McGeer et al., 1988), but any causal relationship has been problematic to evaluate and substantiate.The more recent discovery that microglia and the innate immune system are essential for synaptic pruning was a major demonstration of their ability to impart changes to the neural world around them and suggested that mechanistically analogous processes could contribute to both neurological and psychiatric illnesses (Hong et al., 2016;Sekar et al., 2016;Vainchtein andMolofsky, 2020, Tae-In Kam et al., 2020).Similarly, microglia-derived inflammation may induce astrocytes to adopt neurotoxic functions or to lose neurotrophic or synaptotrophic functionality (Liddelow et al., 2017, Tae-In Kam et al., 2020, Patani et al., 2023).In addition, microglia, play a pivotal role in mediating inflammation and neuronal plasticity after CNS injury (Lull and Block, 2010;Anttila et al., 2017).Some reports suggest beneficial effects of steroid-PAMs on microglial activation and in PD.In cultured microglia stimulated with LPS, progesterone suppresses NF-κB and JNK activation, as well as TNFα and iNOS production (Lei et al., 2014).Progesterone also prevents depression-like behavior in rats injected with 6-OHDA, a model of PD (Casas et al., 2011).ALLO prevents PD-like disease progression and improves cognition when administered orally for two months to this animal model of PD (Nezhadi et al., 2016).Treatment with ALLO improved dopaminergic neurons and motor performance in the MPTP-lesioned mouse model of Parkinson's disease (Adeosun et al., 2012).In contrast, treatment with progesterone did not improve L-dopa induced dyskinesia in a monkey model of PD (Gomez-Mancilla and Bedard, 1992).Furthermore, in a double-blind study, progesterone administration for two weeks induced rather antidopaminergic effect (Strijks et al., 1999).Some reports suggest that the effects of administration of exogenous ALLO may be beneficial or detrimental depending on pattern of administration and dosing regimen.Bengtsson et al., (2020) reviewed the effects of ALLO on Alzheimer's disease pathology and cognitive function and concluded that ALLO given intermittently promotes neurogenesis, decreases AD-related pathology, and improves cognition.In contrast, continuous exposure to ALLO impairs cognition and deteriorates AD pathology.The reasons for these differential effects remain unclear.Thus, our understanding of the effects of steroid-PAMs on neuroinflammation and of the underlying mechanisms is incomplete and further investigation is needed.
There is evidence of an interplay between GABAergic neurotransmission and neuroinflammation, which modulate each other and contribute to the induction of cognitive and motor impairment in hyperammonemia and HE (Cabrera-Pastor et al., 2019;Agusti et al., 2017).In support of this hypothesis, it has been shown that reducing GABAergic neurotransmission reduces neuroinflammation in rats with hyperammonemia and hepatic encephalopathy (Llansola et al., 2024).
Treatment of hyperammonemic rats with the GABA-A receptor antagonist bicuculline reduces neuroinflammation in hippocampus and cerebellum and improves cognitive and motor function (Malaguarnera et al., 2019 and2021).The neurosteroid pregnenolone-sulfate also improves cognitive and motor function in hyperammonemic rats (Gonzalez-Usano et al., 2014).
While the GABA-A receptor is a validated target with several approved agonist drugs, the development of GABA-A receptor antagonists has been hindered by their propensity to induce seizures at doses needed for therapeutic effect and are therefore problematic for human use because they cause seizures.(Williamson et al., 2004;Johnston, 2013).
A novel, safe way to reduce GABAergic neurotransmission would be to use a compound that modulates activation of GABA-A receptors without interfering with direct activation of the receptors by GABA.Golexanolone (GR 3027) is just such a GABA-modulating steroid antagonist (GAMSA) and is in clinical development by Umecrine Cognition AB (Montagnese et al., 2021;Johansson et al., 2018;Bengtsson et al., 2023).Golexanolone reduces GABAergic tone by blocking allosteric potentiation of GABA-A receptors activation by steroid-PAMs such as ALLO in humans (Johansson et al., 2018).Treatment with golexanolone restores motor coordination and cognitive function in hyperammonemic rats, and in rats with portacaval shunts, another model of HE (Mincheva et al., 2022).In humans, golexanolone has been shown to enter the brain and reverse the effects of an ALLO challenge at doses which appear safe and well-tolerated in healthy adults and patients with cirrhosis (Johansson et al., 2018) and improved cognitive performance and certain EEG parameters in a Phase 2a study in cirrhotic patients with minimal HE (Montagnese et al., 2021).
Hyperammonemia is a proven cause of HE in patients with clinically decompensated cirrhosis (Rockey et al., 2014, Vierling et al., 2016), and current evidence suggests that the effect of hyperammonemia is likely mediated by neuroinflammation and enhanced GABA-ergic transmission.It has been shown that golexanolone reduces neuroinflammation in rat models of hyperammonemia and HE.HE is triggered by peripheral inflammation which induces neuroinflammation in the brain, especially the cerebellum (Mincheva et al., 2022).Chronic moderate hyperammonemia was induced by feeding rats an ammonia-containing diet for 6 weeks.From the second week, 50 mg/kg golexanolone or the same volume of a vehicle was given orally daily via gavage.At 6 weeks of hyperammonemia and 5 weeks of golexanolone treatment, rats were sacrificed and neuroinflammation was analyzed.
Hyperammonemic rats showed activation of microglia and astrocytes in hippocampus, associated with impaired short-term and spatial memory.Treatment with golexanolone reversed both microglia and astrocytes activation in hippocampus and normalized short-term and spatial memory (Mincheva et al., 2022).
Hyperammonemia also increased microglia and astrocytes activation in the cerebellum, and this was associated with enhanced activation of the TNFR1-glutaminase-GAT3 and the TNFR1-CCL2-TrkB-KCC2 pathways and impairment of motor coordination and locomotor gait.Golexanolone reversed all these changes, reducing neuroinflammation, normalizing glial activation and improving motor coordination and  (2022).The effects of neuroinflammation on changes in GABA-AR function GABA release and production is discussed in other reviews (Bäckström et al., 2011, Felipo, 2013, Bengtsson et al. 2019, Arenas et al. 2022a,).
These data show that golexanolone reverses neuroinflammation in the cerebellum and hippocampus and restores cognitive and motor function in hyperammonemic rats.However, these are not the only effects of golexanolone.Hyperammonemic rats also show peripheral inflammation with increased plasma levels of the pro-inflammatory TNFα and reduced levels of the anti-inflammatory IL-10.Golexanolone also reversed these changes, normalizing peripheral inflammation.
Another liver disorder, chronic cholestatic liver disease due to primary biliary cholangitis (PBC) shows fatigue and cognitive impairment that reduces their quality of life.In a rodent model of PBC bile duct ligation, (BDL) induces fatigue, impairs memory and motor coordination, and alters locomotor gait.Golexanolone administration improved these cognitive abnormalities, together with improvement of inflammation, neuroinflammation, and decreased GABAergic neurotransmission in the cerebellum.(Arenas et al., 2023) These findings show that the GAMSA effect and direct improvement of neuroinflammation in the brain are not the only mechanisms by which golexanolone can improve neurological function (Bäckstrom et al., 2023).In addition, the reduction of peripheral inflammation may also contribute to reducing neuroinflammation and improve cognitive and motor function.The underlying mechanisms are still unclear (Mincheva et al., 2022).The effects of neuroinflammation and golexanolone in hyperammonemic rats are summarized in Fig. 1.
These and other results suggest that modulation of GABAergic neurotransmission is a promising new therapeutic approach for inflammatory and autoimmune diseases (Wu et al., 2017).The reduction of neuroinflammation by golexanolone would be a consequence of the reduction of GABAergic neurotransmission, as it occurs for bicuculline (Malaguarnera et al., 2019(Malaguarnera et al., , 2021)).Golexanolone has so far demonstrated satisfactory safety and pharmacokinetics and been well tolerated at doses which reverse the effects of an ALLO challenge in healthy adults and improved selected measures of cognitive performance in patients with cirrhosis (Johansson et al., 2018;Montagnese et al., 2021).These findings collectively support the therapeutic potential of golexanolone to improve neurological function in patients with liver disease as well as in other diseases associated with neuroinflammation and enhanced GABAergic neurotransmission.

Fig. 1 .
Fig. 1. Figure shows an example of peripheral inflammation that causes neuroinflammation.In chronic hyperammonemia, an activation of microglia and astrocytes causes neuroinflammation, impairs cognition, and motor function.Inhibition of microglial and astrocyte activation, e.g., by a receptor modulating steroid antagonist, golexanolone, improves peripheral inflammation, neuroinflammation, neurotransmission and cognitive and motor function in hyperammonemic rats (Mincehva et al.(2022).The effects of neuroinflammation on changes in GABA-AR function GABA release and production is discussed in other reviews(Bäckström et al., 2011, Felipo, 2013, Bengtsson et al. 2019, Arenas et al. 2022a,).