Golexanolone, a GABAA receptor modulating steroid antagonist, restores motor coordination and cognitive function in hyperammonemic rats by dual effects on peripheral inflammation and neuroinflammation

Abstract Aims Hyperammonemic rats show peripheral inflammation, increased GABAergic neurotransmission and neuroinflammation in cerebellum and hippocampus which induce motor incoordination and cognitive impairment. Neuroinflammation enhances GABAergic neurotransmission in cerebellum by enhancing the TNFR1‐glutaminase‐GAT3 and TNFR1‐CCL2‐TrkB‐KCC2 pathways. Golexanolone reduces GABAA receptors potentiation by allopregnanolone. This work aimed to assess if treatment of hyperammonemic rats with golexanolone reduces peripheral inflammation and neuroinflammation and restores cognitive and motor function and to analyze underlying mechanisms. Methods Rats were treated with golexanolone and effects on peripheral inflammation, neuroinflammation, TNFR1‐glutaminase‐GAT3 and TNFR1‐CCL2‐TrkB‐KCC2 pathways, and cognitive and motor function were analyzed. Results Hyperammonemic rats show increased TNFα and reduced IL‐10 in plasma, microglia and astrocytes activation in cerebellum and hippocampus, and impaired motor coordination and spatial and short‐term memories. Treating hyperammonemic rats with golexanolone reversed changes in peripheral inflammation, microglia and astrocytes activation and restored motor coordination and spatial and short‐term memory. This was associated with reversal of the hyperammonemia‐enhanced activation in cerebellum of the TNFR1‐glutaminase‐GAT3 and TNFR1‐CCL2‐TrkB‐KCC2 pathways. Conclusion Reducing GABAA receptors activation with golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in hyperammonemic rats. The effects identified would also occur in patients with hepatic encephalopathy and, likely, in other pathologies associated with neuroinflammation.


| INTRODUC TI ON
Several million patients with liver cirrhosis suffer minimal hepatic encephalopathy (MHE), with mild cognitive impairment and motor incoordination, which reduces the quality of life and increases the risk of accidents and hospitalizations, thus imposing heavy costs on health systems. [1][2][3][4][5][6][7] Treatment of MHE may improve cognitive and motor function in MHE patients. Rifaximin is a non-permeable antibiotic approved for preventing hepatic encephalopathy (HE) appearance in cirrhotic patients. 8 Rifaximin treatment improves neurological function in 60% of MHE patients, but not in the remaining 40%. 9 There are no specific treatments for the neurological alterations in MHE and clinical HE and new, more effective treatments, acting on the mechanisms that induce cognitive and motor impairments are needed.
Hyperammonemia and peripheral inflammation induce neurological impairment in patients and animal models. [10][11][12][13][14][15] Hyperammonemic rats reproduce the cognitive and motor alterations of MHE patients. Hyperammonemia induces peripheral inflammation, which induces neuroinflammation. Cognitive impairment is mainly due to neuroinflammation-induced alterations in glutamatergic neurotransmission in hippocampus. [14][15][16][17][18] Motor incoordination is mainly due to neuroinflammation-induced increase in GABAergic neurotransmission in the cerebellum. Hyperammonemia induces microglia and astrocytes activation and increases TNFα and membrane expression of the TNFα receptor TNFR1 in the cerebellum. Increased activation of TNFR1 enhances GABAergic neurotransmission by activating the TNFR1-glutaminase-GAT3 pathway and the TNFR1-CCL2-TrkB-KCC2 pathway. 14,19,20 Enhanced GABAergic neurotransmission induces motor incoordination in hyperammonemia and MHE. A plausible therapeutic approach to improve cognitive and motor function would be treatments aiming at reducing GABAergic neurotransmission. Reducing GABAergic neurotransmission using the GABA A receptor antagonist bicuculline or with the neurosteroid pregnenolone sulfate improves cognitive and motor impairments in hyperammonemic rats. [21][22][23][24][25] However, the use of antagonists of GABA A receptors would not be useful in clinical practice. A more useful approach would be to reduce GABAergic neurotransmission by using compounds that modulate indirectly GABA A receptors. Allopregnanolone (3ahydroxy-5a-pregnane-20-one) is a positive allosteric modulator of GABA A receptors which is increased in the brain of hyperammonemic rats 25 and of cirrhotic patients who died in hepatic coma, 26 and may enhance GABAergic neurotransmission. Reducing the potentiation of GABA A receptors activation by allopregnanolone could be a safe way to reduce GABAergic neurotransmission. Golexanolone (GR3027), a novel investigational drug in clinical development, is a GABA A receptor modulating steroid antagonist which reduces the potentiation of GABA A receptors by allopregnanolone in animal models and in humans and is a promising therapeutic tool to improve cognitive and motor function in hyperammonemia and HE.
Repeated subcutaneous injections of golexanolone, at doses generating plasma levels safe in animal toxicology studies, restores motor coordination and cognitive function in hyperammonemic rats, a model of MHE, 27 and improves cognitive performance in a pilot phase 2a study in patients with MHE. 28 However, the mechanisms involved have not been fully elucidated. 27,28 There is an interplay between GABAergic neurotransmission and neuroinflammation, which modulate each other, and contribute to induction of cognitive and motor impairment. 14,29 It would be therefore possible to reduce neuroinflammation by reducing GABAergic neurotransmission. We hypothesized that the beneficial effects of golexanolone on cognitive and motor function in hyperammonemic rats are not only due to its direct action on GABAergic neurotransmission, but golexanolone would also reduce neuroinflammation, which would contribute to reducing GABAergic neurotransmission by reducing the activation of the TNFR1-glutaminase-GAT3 pathway and the TNFR1-CCL2-TrkB-KCC2 pathway.
Treatment of hyperammonemic rats with bicuculline reduces peripheral inflammation. 21 Golexanolone could also reduce peripheral inflammation, which may contribute to reducing neuroinflammation and improving cognitive and motor function.
The aims of this study were to: (1) assess if golexanolone reduces neuroinflammation in cerebellum and hippocampus of hyperammonemic rats, and if it reduces microglia and/or astrocytes activation; (2) identify mechanisms by which golexanolone may reduce GABAergic neurotransmission in cerebellum: analyze the effects on the TNFR1glutaminase-GAT3 pathway and the TNFR1-CCL2-TrkB-KCC2 pathway; (3) assess if golexanolone improves peripheral inflammation by analyzing TNFα, IL-10 and TGFβ. In our previous study with golexanolone, the compound was administered by subcutaneous injection. 27 As this is not a convenient administration route in clinical practice, we have developed a formulation which may be administered orally.
We also aimed to assess if intragastric administration of golexanolone improves cognitive and motor function in hyperammonemic rats.

| Animal model and treatment
Forty-eight male Wistar rats (220-250 g) were used. Rats were made hyperammonemic by feeding an ammonia-containing diet as made by Felipo et al. 30 The experimental design is shown in Figure 1.
After 1 week of ammonia-containing diet, rats were divided in four groups of twelve rats: controls treated with vehicle (CV), controls treated with golexanolone (CGR); hyperammonemic-vehicle K E Y W O R D S GR3027, inflammation, minimal hepatic encephalopathy, motor incoordination, spatial memory (HV) and hyperammonemic rats treated with golexanolone (HGR).

| Motor function
Motor function: motor coordination and gait parameters was assessed after two weeks of golexanolone administration.

| Footprint analysis of locomotor gait in the CatWalk™
This is a video-based automated gait analysis system (Noldus, Wageningen, The Netherlands). Three trials were recorded each day during two days. Gait analysis values are the mean of six runs. Data were analyzed using the CatWalk analysis software (v 7.1). 31

| Motorater
A kinematic analysis of motor coordination was conducted using MotoRater apparatus (TSE Systems, Germany) as in Ref. [32]. Each day three uninterrupted runs were recorded for each rat, over three days. The runs were analyzed by counting and classifying the steps as correct or wrong paw placements. The results are expressed as percentage of total steps and are the mean of nine runs.

| Novel object recognition (NOR) and novel object location (NOL) memory tests
Tests were performed in an open-field arena with visuospatial cues on the walls as in Ref. [33]. The NOL test was performed on day six. The NOR test was performed on day seven. A discrimination ratio was calculated as the difference between the times spent exploring the object whose location had been changed (NOL) or the new object (NOR) with the unchanged object divided by total time exploring.

| Short-term spatial recognition memory
Short-term spatial recognition memory was analyzed using a Y-maze.
The rat was placed into one arm (start arm) and allowed to explore the maze with one arm closed, for 2 min (training trial) two times, with 1 min of inter-trial interval. Then, the rat was allowed to explore all three arms for 2 min (test trial). The number of entries into and the time spent in each arm were registered and the discrimination ratio

| Analysis of astrocytes and microglia activation
Sections were scanned with an Aperio Versa system (Leica Biosystems, Germany). Fields at 40× magnification were captured using the software ImageScope64; 8-10 images per rat were taken from three different sections of the hippocampus or the cerebellum.
Microglial activation was analyzed by measuring the area of Iba1 stained cells with IpWin 32 software program and astrocytic activation by measuring the GFAP stained area with ImageJ software as in Ref. [34]

| Statistical analysis
Data are expressed as mean SEM. All statistical analyses were performed using GraphPad Prism software v. 9

| RE SULTS
Based on a chronic toxicity study in rats, a dose of 50 mg/kg/day was selected for the assessment of the effects of orally administered golexanolone. At this dosage, systemic exposure is expected to be in a range that is achievable, and has been tested in healthy volunteers and HE-patients. Moreover, no toxicity was observed at 100 mg/kg/ day, which was the highest dose tested.
Microglia was activated in hippocampus of hyperammonemic rats as indicated by a reduced area of Iba1 marked cells. Golexanolone reversed this reduction (231 ± 10 μm 2 in hyperammonemic rats vs. 333 ± 15 μm 2 in hyperammonemic rats treated with golexanolone, F I G U R E 2 Golexanolone treatment reduces peripheral inflammation in hyperammonemic rats. TNF-a in plasma was analyzed by ELISA (A). IL-10 (B) and TGFb (C) in plasma were analyzed by Western blot. Values are the mean ± SEM of 9 rats per group in (A and B) and of 11 rats per group in C. Values significantly different from control rats are indicated by asterisk and from hyperammonemic rats by "a". *p < 0.05; **p < 0.01; a p < 0.05. CGR, control group with golexanolone treatment; CV, control rats with treatment vehicle (CAPMUL); HGR, hyperammonemic rats with golexanolone treatment; HV, hyperammonemic rats with vehicle F I G U R E 3 Golexanolone treatment reduces activation of astrocytes and microglia in hippocampus of hyperammonemic rats. Representative immunohistochemistry images of GFAP staining are shown in (A). The area stained by anti-GFAP was quantified in whole hippocampus in two slides/rat from 6 rats per group. Values are expressed as percentage of stained area in control rats (B). Hippocampal microglia was stained with anti-Iba1. Representative images are shown in (C). Microglia activation was quantified by measuring the area of Iba1 + cells (D). Content of Iba1 was analyzed by western blot in whole hippocampus homogenates. Representative bands and quantification is shown in E. Values are the mean ± SEM of 6 rats per group. Values significantly different from control rats are indicated by asterisk and from hyperammonemic rats by "a". *p < 0.05; aaa p < 0.001. CGR, control group with golexanolone treatment; CV, control rats with treatment vehicle (CAPMUL); HGR, hyperammonemic rats with golexanolone treatment; HV, hyperammonemic rats with vehicle and 290 ± 13 μm 2 in controls) indicating reversal of microglia activation ( Figures 3C,D and S2). Western blot analysis of Iba1 content show that total content of Iba1 is not reduced, but slightly increased, indicating that the reduced area of Iba1 + cells in the hippocampus of hyperammonemic rats is not due to a decrease in Iba1 expression, but is caused by a change in morphology of microglial cells to a more ameboid shape with shorter prolongations, indicating activated state of microglia ( Figure 3E).
We analyzed spatial memory using the novel object location test. The discrimination ratio was reduced in hyperammonemic (−0.067 ± 0.067 vs. 0.14 ± 0.046 in controls) and treatment with golexanolone restored it (0.16 ± 0.046). Short-term spatial memory in the Y maze was impaired in hyperammonemic rats as reflected in the lower discrimination ratio (0.034 ± 0.034 compared with 0.46 ± 0.028 in controls). Golexanolone reversed the impairment of short-term memory returning the discrimination ratio to normal values (0.046 ± 0.027) ( Figure 4B).
We also analyzed glial activation in cerebellum. Hyperammonemic rats showed astrocytes activation, as shown by immunohistochemistry by the increased (129 ± 5%) area stained by GFAP ( Figures 5A,B and S3) and by the increased (130 ± 5%) GFAP content as analyzed by Western blot ( Figure 5C). Golexanolone reversed the increase in astrocytes activation, as indicated by the reversal of the increase in GFAP staining ( Figure 5A,B) and content ( Figure 5C).  (Figures 5D-G, S4, and S5). As occurs in the hippocampus, the total content of Iba1 in the whole cerebellum as analyzed by Western blot was not reduced ( Figure S5H9): The area of Iba1 + cells is reduced due to acquisition of a more ameboid shape due to activation ( Figure 5H).
These results support the idea that golexanolone reduces GABAergic neurotransmission in cerebellum of hyperammonemic rats by different mechanisms. We assessed if this is associated with F I G U R E 4 Golexanolone treatment improves novel object location and short-term memory in hyperammonemic rats. Discrimination ratio was calculated as indicated in methods for novel object location memory (OLM) (A) and for short-term memory in the Y Maze (B) Values are the mean ± SEM of 10 rats per group for OLM and 11 rats per group for short-term memory. Values significantly different from control rats are indicated by asterisk and from hyperammonemic rats by "a". *p < 0.05; a p < 0.05. CGR, control group with golexanolone treatment; CV, control rats with treatment vehicle (CAPMUL); HGR, hyperammonemic rats with golexanolone treatment; HV, hyperammonemic rats with vehicle improvement of motor coordination and function. Hyperammonemic rats show motor incoordination, with increased number of slips in the motorater (1.4 ± 0.16 slips for hyperammonemic rats compared to 0.67 ± 0.12 slips in controls, p = 0.028). Treatment with golexanolone reversed motor incoordination, reducing the number of slips to normal values (0.7 ± 0.14 slips, p = 0.038 compared with hyperammonemic rats without treatment) ( Figure 8A).
Motor function was also assessed in the CatWalk test, which allows an accurate analysis of locomotor gait and detection of different types of gait alterations that reflect impaired fine motor control, modulated by cerebellum. Hyperammonemic rats show increased stride length (13.5 ± 0.07 cm in hyperammonemic rats, compared to 12 ± 0.09 cm in controls, p < 0.0001) which is reversed by golexanolone (12.4 ± 0.06 cm, p = 0.0097 compared with non-treated hyperammonemic rats) ( Figure 8B). Other parameters such as step cycle ( Figure 8C) and print positions ( Figure 8D) show reduced values in hyperammonemic rats and were normalized by golexanolone ( Figure 8C,D). Other parameters of gait affected in hyperammonemia and the effects on them of golexanolone are shown in Table 1.

| DISCUSS ION
The results reported show that treatment with golexanolone reduces peripheral inflammation and reverses the activation of microglia and astrocytes and neuroinflammation in hippocampus and cerebellum of hyperammonemic rats. This is associated with reversal of cognitive impairment and of the alterations in motor function and F I G U R E 5 Golexanolone treatment reduces activation of astrocytes and microglia in cerebellum of hyperammonemic rats. Representative immunohistochemistry images of GFAP staining are shown in (A). The area stained by anti-GFAP was quantified in cerebellum using two slides/rat from 6 rats per group. Values are expressed as percentage of stained area in control rats (B). GFAP content in whole cerebellum was also analyzed by Western blot and expressed as percentage of control rats (C). Representative images of microglia stained with Iba1 in white matter of cerebellum are shown in (D) and in the molecular layer in (F). Microglia activation was quantified by measuring the area of Iba1 + cells in white matter (E) and molecular layer (G). Content of Iba1 was analyzed by western blot in whole cerebellar homogenates. Representative bands and quantification is shown in H. Values are the mean ± SEM of 6 rats per group. Values significantly different from control rats are indicated by asterisk and from hyperammonemic rats by "a". *p < 0.05; **p < 0.01; a p < 0.05; aa p < 0.01 and aaa p < 0.001. CGR, control group with golexanolone treatment; CV, control rats with treatment vehicle (CAPMUL); HGR, hyperammonemic rats with golexanolone treatment; HV, hyperammonemic rats with vehicle coordination. Moreover, we unveil some mechanisms by which this reduction in neuroinflammation by golexanolone may contribute to reduce GABAergic neurotransmission, leading to improved cognitive and motor function.
Golexanolone is a GABA A receptor modulating steroid antagonist that reduces GABAergic neurotransmission by reducing the potentiation by allopregnanolone of GABA A receptors activation. 27,[38][39][40] However, we show here that this is not the only mechanism by which golexanolone may reduce the enhanced GABAergic neurotransmission in hyperammonemic rats and improve neurological function.
Golexanolone reduces peripheral inflammation in hyperammonemic rats, normalizing TNFα and IL-10 levels. Chronic hyperammonemia is enough to induce peripheral inflammation, which mediates the induction of neuroinflammation in hippocampus and cognitive impairment. 15 Treatment of hyperammonemic rats with bicuculline, an antagonist of GABA A receptors, also normalizes plasma levels of TNFα and IL-10. 21 We show here that reducing GABAergic have been proposed, [42][43][44][45][46][47][48][49] suggesting that GABAergic components are a new therapeutic approach for inflammatory and autoimmune diseases. 50 The results reported here show that treatment with golexanolone reverses peripheral inflammation in hyperammonemic rats and could be beneficial in certain inflammatory or autoimmune diseases.

F I G U R E 6
Golexanolone treatment reverses the changes in the TNFα-TNFR1-GAT3 pathway induced by hyperammonemia in cerebellum. The content of TNFα (A), glutaminase (C) and GAT3 (D) were analyzed by Western blot. Membrane expression of TNFR1 (B) and of GAT3 (E) were analyzed using BS3 crosslinker and Western blot. Values are the mean ± SEM of 6 rats per group. Values significantly different from control rats are indicated by asterisk and from hyperammonemic rats by "a" and from control rats treated with golexanolone by "b". *p < 0.05; a p < 0.05; bb p < 0.01. CGR, control group with golexanolone treatment; CV, control rats with treatment vehicle (CAPMUL); HGR, hyperammonemic rats with golexanolone treatment; HV, hyperammonemic rats with vehicle We show here that golexanolone reduces both peripheral inflammation and neuroinflammation, thus inducing antiinflammatory effects. Antiinflammatory effects have been also reported for allopregnanolone, which reduces the proinflammatory signaling induced by activation of TLR4. 51 However, these effects are independent of GABA A receptors, and are also induced by pregnenolone, which does not potentiate GABA A receptors activation, and are due to the steroid D ring. This antiinflammatory effect would be due to interference by allopregnanolone of MyD88 binding to TLRs. 52 Allopregnanolone reverses neurogenic and cognitive deficits in mouse models of Alzheimer's disease. 53,54 These effects are mediated by activation of GABA A receptors 53,55 indicating that they are not due to the antiinflammatory effect of allopregnanolone. 51,52 Allopregnanolone induces biphasic effects, 56 depending on the persistence of the increase of allopregnanolone, single or intermittent exposure-induced beneficial effects on neurogenesis and learning, 56 while chronically elevated allopregnanolone levels impaired learning. [57][58][59] These effects would be a consequence of the persistence of the activation and do not seem to depend on the concentration of allopregnanolone, as supported by the data on Table 1 in Ref. [55] summarizing many studies using chronic or acute administration of different doses of allopregnanolone 56 Allopregnanolone levels are increased in brain of cirrhotic patients who died in hepatic coma, 26 and in rats with hepatic encephalopathy due to portacaval shunts. 60 Treatment of these rats with indomethacin reduces allopregnanolone in brain and improves locomotor deficits. 60 Allopregnanolone is also increased in brain of hyperammonemic rats. 25  Values significantly different from control rats are indicated by asterisk and from hyperammonemic rats by "a". *p < 0.05; **p < 0.01; a p < 0.05; aaaa p < 0.0001. CGR, control group with golexanolone treatment; CV, control rats with treatment vehicle (CAPMUL); HGR, hyperammonemic rats with golexanolone treatment; HV, hyperammonemic rats with vehicle values the TNFα content, membrane expression of TNFR1 and the content and membrane expression of GAT3. This would reverse the increase in extracellular GABA and its contribution to enhanced GABAergic neurotransmission.
Another mechanism by which increased activation of TNFR1 enhances GABAergic neurotransmission is by activating the TNFR1-CCL2-TrkB-KCC2 pathway. Enhanced activation of TNFR1 increases CCL2 content, which contributes to activate microglia and increase BDNF, which activates TrkB that increases membrane expression of the chloride co-transporter KCC2. KCC2 is expressed in neurons and extrudes chloride ion from the neuron increasing the chloride gradient, hyperpolarizing the neuron and increasing the responses to activation of GABA A receptors by allopregnanolone and GABA. 19,[62][63][64] We show here that, in cerebellum of hyperammonemic rats, golexanolone also reverses the overactivation of the TNFR1-CCL2-TrkB-KCC2 pathway, which would normalize the transmembrane chloride gradient, contributing to reduce GABAergic neurotransmission.
Golexanolone also reduces GABAergic neurotransmission by reversing the increase of the GABA synthesizing GAD67, which would reduce GABA concentration, and by reversing the increased membrane expression of the β3 subunit of GABA A receptors, which would reduce its activation.  Note: Footprint of gait in rats was analyzed in the Catwalk™, analyzing different parameters characterizing locomotor gait, both related to paw statistics and of step sequence, the last assessing fine motor coordination. Values are the mean ± SEM of 9 rats per group. Statistical analysis with One-way ANOVA brought the following data: Abbreviations: CV, control rats with treatment vehicle (CAPMUL); CGR, control group with golexanolone treatment; HV, hyperammonemic rats with vehicle; HGR, hyperammonemic rats with golexanolone treatment.
* p < 0.05; ** p < 0.01; a p < 0.05. activation 65 enhanced GABAergic neurotransmission in cerebellum, 66 and impaired cognitive function and motor function and coordination. [1][2][3][4]7,12,67 In addition to the expected effects in HE, golexanolone may also have beneficial effects in improving neurological function in patients with other pathologies associated with neuroinflammation and enhanced GABAergic neurotransmission. It has been shown that oral administration of golexanolone (GR3027) mitigates inhibition of brain function induced by allopregnanolone in healthy adult males at doses which are clinically well tolerated. 39 Moreover, safety, pharmacokinetics and efficacy of golexanolone has been investigated in adult patients with cirrhosis with promising results. Golexanolone exhibited satisfactory safety and pharmacokinetic, was well tolerated and associated with improvement in cognitive performance. 28 These studies, together with the data reported here support the therapeutic potential of golexanolone.

| CON CLUS ION
Reducing GABA A receptors activation with golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in hyperammonemic rats. The mechanistic and therapeutic effects identified would also occur in patients with hepatic encephalopathy and, likely, in other pathologies associated with neuroinflammation.

ACK N OWLED G M ENTS
Funding support has been covered by Umecrine Cognition AB.

CO N FLI C T O F I NTE R E S T
This study was financed by Umecrine Cognition AB, which is developing GR3027/Golexanolone.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.