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

Abstract

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.

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

“…Slices were transferred to incubation wells in a perfusion system (Campden Instruments, Model 7450) and incubated for 15 min at 35.5 °C in Krebs buffer for stabilization. Once stabilized, the slices from hyperammonemic rats were incubated during 30 min at 35.5 °C with the following treatments, all of them dissolved in Krebs buffer and aerated: 1 ng/mL of recombinant TGFβ (Miltenyi Biotec) (HA + rec TGFβ) [ 62 ], 1.2 µg/mL of anti-TGFβ antibody (Abcam) (HA + T), 10 µg/mL of extracellular vesicles from MSCs (HA + EVs) [ 43 ], 10 µg/mL of extracellular vesicles from MSCs previously treated with 1.2 µg/mL of anti-TGFβ antibody (Abcam) for 1 h at 37 °C (HA + EVs + anti-TGFβ) [ 83 ], 10 µg/mL of extracellular vesicles from MSCs lacking TGFβ as described above (HA + EVs lacking TGFβ; 10 µg/mL of extracellular vesicles from MSCs plus 2 µg/mL of galunisertib, an antagonist of TGFβ receptor (HA + EVs + anti-TGFβR [ 10 , 56 ]. All treatments were for 30 min.…”

Contribution of extracellular vesicles to neuroinflammation and cognitive and motor deficits in hyperammonemia and hepatic encephalopathy

“…More recently, extracellular vesicles (EVs) have been suggested as an alternative mechanism for communication between the periphery and the brain in HE [2,[11][12][13] . The main reasons to study the contribution of EVs in the induction of neuroinflammation and the subsequent cognitive and motor impairment in HE are their ability to trespass the blood-brain-barrier (BBB) and the results reported in previous studies pointing out their relevance as intercellular mediators in different pathologies associated with chronic inflammation, including neurological diseases.…”

“…Izquierdo-Altarejos et al demonstrated for the first time that intravenously injected HA-EVs reached the cerebellum of control rats, co-localizing with Purkinje neurons and microglia [11] . HA-EVs induced neuroinflammation in cerebellum, with microglia and astrocyte activation, and lead to motor incoordination in control rats.…”

Bidirectional Communication Between the Brain and Other Organs: The Role of Extracellular Vesicles

“…Plasma EVs in hyperammonemic rats were changed and the TNF-α level was elevated. When injected into normal rats, EVs from plasma of hyperammonemic rats reached the cerebellum and caused changes in Purkinje neurons and microglia in particular particularly Purkinje neurons and microglia, further resulting in neuroinflammation and motor incoordination (Izquierdo-Altarejos et al 2022 ). Also, elevated TNF-α levels in EVs from hyperammonemic rats' blood activate the TNF receptor 1 (TNFR1)/C–C motif chemokine ligand 2 (CCL2)/BDNF/tropomyosin receptor kinase B (TrkB)/KCC2 and TNFR1/NF-kB/glutaminase/GABA transporter 3 (GAT3) pathways, thereby enhancing cerebellar GABAergic neurotransmission.…”

“…Also, elevated TNF-α levels in EVs from hyperammonemic rats' blood activate the TNF receptor 1 (TNFR1)/C–C motif chemokine ligand 2 (CCL2)/BDNF/tropomyosin receptor kinase B (TrkB)/KCC2 and TNFR1/NF-kB/glutaminase/GABA transporter 3 (GAT3) pathways, thereby enhancing cerebellar GABAergic neurotransmission. As a result, microglia activation, increased GABAergic neurotransmission, and motor incoordination occur (Izquierdo-Altarejos et al 2022 ). Apolipoprotein E4 (ApoE4) alleles are strongly associated with age-related cognitive impairment and AD (Kanekiyo et al 2014 ; Strittmatter et al 1993 ).…”