Search

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 TGFβ levels and membrane expression of TGFβ receptors in hippocampus. This leads to microglia activation and reduced Smad7–IkB pathway, which induces NF-κB nuclear translocation in neurons, increasing IL-1β which alters AMPA and NMDA receptors membrane expression, leading to cognitive impairment. These effects are reversed by TGFβ in the EVs from MSCs, which activates TGFβ receptors, reducing microglia activation and NF-κB nuclear translocation in neurons by normalizing the Smad7–IkB pathway. This normalizes IL-1β, 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., This work was supported in part by the Ministerio de Ciencia e Innovación Spain (PID2020-113388RB-I00), Consellería Educación Generalitat Valenciana (PROMETEOII/2018/051; CIPROM2021/082), and co-funded with European Regional Development Funds (ERDF). PIA has a contract from Ministerio de Ciencia, Innovación y Universidades (FPU17/01698) and ACP from Instituto de Salud Carlos III (Postdoctoral Sara Borrell, CD17/00031).

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 GABARats 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.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.Reducing GABA

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.

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, TNF&alpha, 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.

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.