Blocking glycine receptors reduces neuroinflammation and restores neurotransmission in cerebellum through ADAM17-TNFR1-NF-κβ pathway

BACKGROUND: Chronic hyperammonemia induces neuroinflammation in cerebellum, with glial activation and enhanced activation of the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway. Hyperammonemia also increases glycinergic neurotransmission. These alterations contribute to cognitive and motor impairment. Activation of glycine receptors is reduced by extracellular cGMP, which levels are reduced in cerebellum of hyperammonemic rats in vivo. We hypothesized that enhanced glycinergic neurotransmission in hyperammonemic rats (1) contributes to induce neuroinflammation and glutamatergic and GABAergic neurotransmission alterations; (2) is a consequence of the reduced extracellular cGMP levels. The aims were to assess, in cerebellum of hyperammonemic rats, (a) whether blocking glycine receptors with the antagonist strychnine reduces neuroinflammation; (b) the cellular localization of glycine receptor; (c) the effects of blocking glycine receptors on the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway and microglia activation; (d) whether adding extracellular cGMP reproduces the effects of strychnine. METHODS: We analyzed in freshly isolated cerebellar slices from control or hyperammonemic rats the effects of strychnine on activation of microglia and astrocytes, the content of TNFa and IL1b, the surface expression of ADAM17, TNFR1 and transporters, the phosphorylation levels of ERK, p38 and ADAM17. The cellular localization of glycine receptor was assessed by immunofluorescence. We analyzed the content of TNFa, IL1b, HMGB1, glutaminase, and the level of TNF-a mRNA and NF-κB in Purkinje neurons. Extracellular concentrations of glutamate and GABA were performed by in vivo microdialysis in cerebellum. We tested whether extracellular cGMP reproduces the effects of strychnine in ex vivo cerebellar slices. RESULTS: Glycine receptors are expressed mainly in Purkinje cells. In hyperammonemic rats, enhanced glycinergic neurotransmission leads to reduced membrane expression of ADAM17, resulting in increased surface expression and activation of TNFR1 and of the associated NF-kB pathway. This increases the expression in Purkinje neurons of TNFa, IL-1b, HMGB1, and glutaminase. Increased glutaminase activity leads to increased extracellular glutamate, which increases extracellular GABA. Increased extracellular glutamate and HMGB1 potentiate microglial activation. Blocking glycine receptors with strychnine or extracellular cGMP completely prevents the above pathway in hyperammonemic rats. CONCLUSIONS: Glycinergic neurotransmission modulates neuroinflammation. Enhanced glycinergic neurotransmission in hyperammonemia would be due to reduced extracellular cGMP. These results shed some light on possible new therapeutic target pathways for pathologies associated to neuroinflammation.