Neuroinflammation alters GABAergic neurotransmission in hyperammonemia and hepatic encephalopathy, leading to motor incoordination. Mechanisms and therapeutic implications.

Enhanced GABAergic neurotransmission contributes to impairment of motor coordination and gait and of cognitive function in different pathologies, including hyperammonemia and hepatic encephalopathy. Neuroinflammation is a main contributor to enhancement of GABAergic neurotransmission through increased activation of different pathways. For example, enhanced activation of the TNFα-TNFR1-NF-κB-glutaminase-GAT3 pathway and the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway in cerebellum of hyperammonemic rats enhances GABAergic neurotransmission. This is mediated by mechanisms affecting GABA synthesizing enzymes GAD67 and GAD65, total and extracellular GABA levels, membrane expression of GABAA receptor subunits, of GABA transporters GAT1 and GAT three and of chloride co-transporters. Reducing neuroinflammation reverses these changes, normalizes GABAergic neurotransmission and restores motor coordination. There is an interplay between GABAergic neurotransmission and neuroinflammation, which modulate each other and altogether modulate motor coordination and cognitive function. In this way, neuroinflammation may be also reduced by reducing GABAergic neurotransmission, which may also improve cognitive and motor function in pathologies associated to neuroinflammation and enhanced GABAergic neurotransmission such as hyperammonemia, hepatic encephalopathy or Parkinson's disease. This provides therapeutic targets that may be modulated to improve cognitive and motor function and other alterations such as fatigue in a wide range of pathologies. As a proof of concept it has been shown that antagonists of GABAA receptors such as bicuculline reduces neuroinflammation and improves cognitive and motor function impairment in rat models of hyperammonemia and hepatic encephalopathy. Antagonists of GABAA receptors are not ideal therapeutic tools because they can induce secondary effects. As a more effective treatment to reduce GABAergic neurotransmission new compounds modulating it by other mechanisms are being developed. Golexanolone reduces GABAergic neurotransmission by reducing the potentiation of GABAA receptor activation by neurosteroids such as allopregnanolone. Golexanolone reduces neuroinflammation and GABAergic neurotransmission in animal models of hyperammonemia, hepatic encephalopathy and cholestasis and this is associated with improvement of fatigue, cognitive impairment and motor incoordination. This type of compounds may be useful therapeutic tools to improve cognitive and motor function in different pathologies associated with neuroinflammation and increased GABAergic neurotransmission.

Golexanolone improves fatigue, motor incoordination and gait and memory in rats with bile duct ligation.

BACKGROUND AND AIMS: Many patients with the chronic cholestatic liver disease primary biliary cholangitis (PBC) show fatigue and cognitive impairment that reduces their quality of life. Likewise, rats with bile duct ligation (BDL) are a model of cholestatic liver disease. Current PBC treatments do not improve symptomatic alterations such as fatigue or cognitive impairment and new, more effective treatments are therefore required. Golexanolone reduces the potentiation of GABAA receptors activation by neurosteroids. Golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in rats with chronic hyperammonemia. The aims of the present study were to assess if golexanolone treatment improves fatigue and cognitive and motor function in cholestatic BDL rats and if this is associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. METHODS: Rats were subjected to bile duct ligation. One week after surgery, oral golexanolone was administered daily to BDL and sham-operated controls. Fatigue was analysed in the treadmill, motor coordination in the motorater, locomotor gait in the Catwalk, and short-term memory in the Y-maze. We also analysed peripheral inflammation, neuroinflammation, and GABAergic neurotransmission markers by immunohistochemistry and Western blot. RESULTS: BDL induces fatigue, impairs memory and motor coordination, and alters locomotor gait in cholestatic rats. Golexanolone improves these alterations, and this was associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. CONCLUSION: Golexanolone may have beneficial effects to treat fatigue, and motor and cognitive impairment in patients with the chronic cholestatic liver disease PBC.

Enhanced Activation of the S1PR2-IL-1ß-Src-BDNF-TrkB Pathway Mediates Neuroinflammation in the Hippocampus and Cognitive Impairment in Hyperammonemic Rats.

Hyperammonemia contributes to hepatic encephalopathy. In hyperammonemic rats, cognitive function is impaired by altered glutamatergic neurotransmission induced by neuroinflammation. The underlying mechanisms remain unclear. Enhanced sphingosine-1-phosphate receptor 2 (S1PR2) activation in the cerebellum of hyperammonemic rats contributes to neuroinflammation. in In hyperammonemic rats, we assessed if blocking S1PR2 reduced hippocampal neuroinflammation and reversed cognitive impairment and if the signaling pathways were involved. S1PR2 was blocked with intracerebral JTE-013, and cognitive function was evaluated. The signaling pathways inducing neuroinflammation and altered glutamate receptors were analyzed in hippocampal slices. JTE-013 improved cognitive function in the hyperammonemic rats, and hyperammonemia increased S1P. This increased IL-1ß, which enhanced Src activity, increased CCL2, activated microglia and increased the membrane expression of the NMDA receptor subunit GLUN2B. This increased p38-MAPK activity, which altered the membrane expression of AMPA receptor subunits and increased BDNF, which activated the TrkB → PI3K → Akt → CREB pathway, inducing sustained neuroinflammation. This report unveils key pathways involved in the induction and maintenance of neuroinflammation in the hippocampus of hyperammonemic rats and supports S1PR2 as a therapeutic target for cognitive impairment.

Hepatic encephalopathy: investigational drugs in preclinical and early phase development.

INTRODUCTION: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome, in patients with liver disease, which affects life quality and span. Current treatments are lactulose or rifaximin, acting on gut microbiota. Treatments aiming ammonia levels reduction have been tested with little success. AREAS COVERED: Pre-clinical research shows that the process inducing HE involves sequentially: liver failure, altered microbiome, hyperammonemia, peripheral inflammation, changes in immunophenotype and extracellular vesicles and neuroinflammation, which alters neurotransmission impairing cognitive and motor function. HE may be reversed using drugs acting at any step: modulating microbiota with probiotics or fecal transplantation; reducing peripheral inflammation with anti-TNFα, autotaxin inhibitors or silymarin; reducing neuroinflammation with sulforaphane, p38 MAP kinase or phosphodiesteras 5 inhibitors, antagonists of sphingosine-1-phosphate receptor 2, enhancing meningeal lymphatic drainage or with extracellular vesicles from mesenchymal stem cells; reducing GABAergic neurotransmission with indomethacin or golexanolone. EXPERT OPINION: A factor limiting the progress of HE treatment is the lack of translation of research advances into clinical trials. Only drugs acting on microbiota or ammonia reduction have been tested in patients. It is urgent to change the mentality on how to approach HE treatment to develop clinical trials to assess drugs acting on the immune system/peripheral inflammation, neuroinflammation or neurotransmission to improve HE.

Extracellular vesicles from hyperammonemic rats induce neuroinflammation in hippocampus and impair cognition in control rats.

Patients with liver cirrhosis show hyperammonemia and peripheral inflammation and may show hepatic encephalopathy with cognitive impairment, reproduced by rats with chronic hyperammonemia. Peripheral inflammation induces neuroinflammation in hippocampus of hyperammonemic rats, altering neurotransmission and leading to cognitive impairment. Extracellular vesicles (EVs) may transmit pathological effects from the periphery to the brain. We hypothesized that EVs from peripheral blood would contribute to cognitive alterations in hyperammonemic rats. The aims were to assess whether EVs from plasma of hyperammonemic rats (HA-EVs) induce cognitive impairment and to identify the underlying mechanisms. Injection of HA-EVs impaired learning and memory, induced microglia and astrocytes activation and increased TNFα and IL-1ß. Ex vivo incubation of hippocampal slices from control rats with HA-EVs reproduced these alterations. HA-EVs increased membrane expression of TNFR1, reduced membrane expression of TGFßR2 and Smad7 and IκBα levels and increased IκBα phosphorylation. This led to increased activation of NF-κB and IL-1ß production, altering membrane expression of NR2B, GluA1 and GluA2 subunits, which would be responsible for cognitive impairment. All these effects of HA-EVs were prevented by blocking TNFα, indicating that they were mediated by enhanced activation of TNFR1 by TNFα. We show that these mechanisms are very different from those leading to motor incoordination, which is due to altered GABAergic neurotransmission in cerebellum. This demonstrates that peripheral EVs play a key role in the transmission of peripheral alterations to the brain in hyperammonemia and hepatic encephalopathy, inducing neuroinflammation and altering neurotransmission in hippocampus, which in turn is responsible for the cognitive deficits.

Extracellular vesicles from mesenchymal stem cells reduce neuroinflammation in hippocampus and restore cognitive function in hyperammonemic rats.

Chronic hyperammonemia, a main contributor to hepatic encephalopathy (HE), leads to neuroinflammation which alters neurotransmission leading to cognitive impairment. There are no specific treatments for the neurological alterations in HE. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) reduce neuroinflammation in some pathological conditions. The aims were to assess if treatment of hyperammonemic rats with EVs from MSCs restores cognitive function and analyze the underlying mechanisms. EVs injected in vivo reach the hippocampus and restore performance of hyperammonemic rats in object location, object recognition, short-term memory in the Y-maze and reference memory in the radial maze. Hyperammonemic rats show reduced TGFß levels and membrane expression of TGFß receptors in hippocampus. This leads to microglia activation and reduced Smad7-IkB pathway, which induces NF-κB nuclear translocation in neurons, increasing IL-1ß which alters AMPA and NMDA receptors membrane expression, leading to cognitive impairment. These effects are reversed by TGFß in the EVs from MSCs, which activates TGFß receptors, reducing microglia activation and NF-κB nuclear translocation in neurons by normalizing the Smad7-IkB pathway. This normalizes IL-1ß, AMPA and NMDA receptors membrane expression and, therefore, cognitive function. EVs from MSCs may be useful to improve cognitive function in patients with hyperammonemia and minimal HE.

Extracellular Vesicles From Hyperammonemic Rats Induce Neuroinflammation in Cerebellum of Normal Rats: Role of Increased TNFα Content.

Hyperammonemia plays a main role in the neurological impairment in cirrhotic patients with hepatic encephalopathy. Rats with chronic hyperammonemia reproduce the motor incoordination of patients with minimal hepatic encephalopathy, which is due to enhanced GABAergic neurotransmission in cerebellum as a consequence of neuroinflammation. Extracellular vesicles (EVs) could play a key role in the transmission of peripheral alterations to the brain to induce neuroinflammation and neurological impairment in hyperammonemia and hepatic encephalopathy. EVs from plasma of hyperammonemic rats (HA-EVs) injected to normal rats induce neuroinflammation and motor incoordination, but the underlying mechanisms remain unclear. The aim of this work was to advance in the understanding of these mechanisms. To do this we used an ex vivo system. Cerebellar slices from normal rats were treated ex vivo with HA-EVs. The aims were: 1) assess if HA-EVs induce microglia and astrocytes activation and neuroinflammation in cerebellar slices of normal rats, 2) assess if this is associated with activation of the TNFR1-NF-kB-glutaminase-GAT3 pathway, 3) assess if the TNFR1-CCL2-BDNF-TrkB pathway is activated by HA-EVs and 4) assess if the increased TNFα levels in HA-EVs are responsible for the above effects and if they are prevented by blocking the action of TNFα. Our results show that ex vivo treatment of cerebellar slices from control rats with extracellular vesicles from hyperammonemic rats induce glial activation, neuroinflammation and enhance GABAergic neurotransmission, reproducing the effects induced by hyperammonemia in vivo. Moreover, we identify in detail key underlying mechanisms. HA-EVs induce the activation of both the TNFR1-CCL2-BDNF-TrkB-KCC2 pathway and the TNFR1-NF-kB-glutaminase-GAT3 pathway. Activation of these pathways enhances GABAergic neurotransmission in cerebellum, which is responsible for the induction of motor incoordination by HA-EVs. The data also show that the increased levels of TNFα in HA-EVs are responsible for the above effects and that the activation of both pathways is prevented by blocking the action of TNFα. This opens new therapeutic options to improve motor incoordination in hyperammonemia and also in cirrhotic patients with hepatic encephalopathy and likely in other pathologies in which altered cargo of extracellular vesicles contribute to the propagation of the pathology.

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

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.

Extracellular Vesicles from Hyperammonemic Rats Induce Neuroinflammation and Motor Incoordination in Control Rats.

Minimal hepatic encephalopathy is associated with changes in the peripheral immune system which are transferred to the brain, leading to neuroinflammation and thus to cognitive and motor impairment. Mechanisms by which changes in the immune system induce cerebral alterations remain unclear. Extracellular vesicles (EVs) seem to play a role in this process in certain pathologies. The aim of this work was to assess whether EVs play a role in the induction of neuroinflammation in cerebellum and motor incoordination by chronic hyperammonemia. We characterized the differences in protein cargo of EVs from plasma of hyperammonemic and control rats by proteomics and Western blot. We assessed whether injection of EVs from hyperammonemic to normal rats induces changes in neuroinflammation in cerebellum and motor incoordination similar to those exhibited by hyperammonemic rats. We found that hyperammonemia increases EVs amount and alters their protein cargo. Differentially expressed proteins are mainly associated with immune system processes. Injected EVs enter Purkinje neurons and microglia. Injection of EVs from hyperammonemic, but not from control rats, induces motor incoordination, which is mediated by neuroinflammation, microglia and astrocytes activation and increased IL-1b, TNFα, its receptor TNFR1, NF-kB in microglia, glutaminase I, and GAT3 in cerebellum. Plasma EVs from hyperammonemic rats carry molecules necessary and sufficient to trigger neuroinflammation in cerebellum and the mechanisms leading to motor incoordination.

Peripheral inflammation induces neuroinflammation that alters neurotransmission and cognitive and motor function in hepatic encephalopathy: Underlying mechanisms and therapeutic implications.

Several million patients with liver cirrhosis suffer minimal hepatic encephalopathy (MHE), with mild cognitive and coordination impairments that reduce their quality of life and life span. Hyperammonaemia and peripheral inflammation act synergistically to induce these neurological alterations. We propose that MHE appearance is because of the changes in peripheral immune system, which are transmitted to brain, leading to neuroinflammation that alters neurotransmission leading to cognitive and motor alterations. We summarize studies showing that MHE in cirrhotic patients is associated with alterations in the immune system and that patients died with HE show neuroinflammation in cerebellum, with microglial and astrocytic activation and Purkinje cell loss. We also summarize studies in animal models of MHE on the role of peripheral inflammation in neuroinflammation induction, how neuroinflammation alters neurotransmission and how this leads to cognitive and motor alterations. These studies identify therapeutic targets and treatments that improve cognitive and motor function. Rats with MHE show neuroinflammation in hippocampus and altered NMDA and AMPA receptor membrane expression, which impairs spatial learning and memory. Neuroinflammation in cerebellum is associated with altered GABA transporters and extracellular GABA, which impair motor coordination and learning in a Y maze. These alterations are reversed by treatments that reduce peripheral inflammation (anti-TNFα, ibuprofen), neuroinflammation (sulphoraphane, p38 inhibitors), GABAergic tone (bicuculline, pregnenolone sulphate) or increase extracellular cGMP (sildenafil or cGMP). The mechanisms identified would also occur in other chronic diseases associated with inflammation, aging and some mental and neurodegenerative diseases. Treatments that improve MHE may also be beneficial to treat these pathologies.

The influence of HE history, HE status and neuropsychological test type on learning ability in patients with cirrhosis.

BACKGROUND & AIMS: Learning ability may be impaired in patients with a history of overt hepatic encephalopathy (OHE). The aim of this study was to compare performance on the first/second attempt at a series of tests. METHODS: Two hundred and fourteen patients with cirrhosis were enrolled. On the day of study, 41% were classed as unimpaired, 38% as having minimal HE and 21% as having mild OHE; 58% had a history of OHE. Performance was compared between two versions of the trail-making test A (TMT-A), and between the first/second half of a simple/choice reaction time (sRT and cRT), and a working memory test (ScanRT). RESULTS: Both patients with and without OHE history improved in TMT-A, sRT and ScanRT. Only patients with no OHE history improved in cRT. All patients, regardless of their HE status on the day of study, improved in TMT-A and sRT. Only patients with mild OHE on the day of study improved in cRT. Only unimpaired patients improved in ScanRT. When OHE history and HE status on the day of study were tested together, only HE status had an effect. The same held true when age, the Model for End Stage Liver Disease (MELD) and educational attainment were adjusted for. CONCLUSIONS: HE status on the day of study and the type of neuropsychological test had an effect on learning ability in a well-characterized group of patients with cirrhosis.