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 GABA_A 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 GABA_A 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.

Keywords: GR3027; inflammation; minimal hepatic encephalopathy; motor incoordination; spatial memory.

FIGURE 1

Scheme showing the experimental design

FIGURE 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

FIGURE 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

FIGURE 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

FIGURE 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

FIGURE 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

FIGURE 7

Golexanolone treatment reverses the changes in the TNFR1‐CCL2‐TrkB‐KCC2 pathway and in β3 subunit of GABA_A receptors and GAD67 induced by hyperammonemia in cerebellum. The content of CCL2 (A), TrkB (C), β3 subunit of GABA_A receptors (E) and GAD67 (F) were analyzed by Western blot. Membrane expression of P2X4 (B) and KCC2 (D) 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”. *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

FIGURE 8

Golexanolone treatment reverses the impairment of motor coordination in the Motorater and motor function in the CatWalk in hyperammonemic rats. Motor coordination was assessed in the Motorater by analyzing wrong foot placements (slips) when the rat crossed a ladder (A). Footprint of locomotor gait was analyzed in the Catwalk™, analyzing the following parameters characterizing locomotor gait: (B) Stride length, (C) Step cycle and (D) Print positions. Values are the mean ± SEM of 11 rats per group in A–C and 9 rats per group in D. 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