Inactivation of gsk3β by ser389 phosphorylationbrain regulation and role in neuroimmunity

Resumen

GSK3 activity needs a tight regulation since this kinase plays crucial roles in processes as development, energy metabolism and immunity. Although GSK3 inhibition by Akt-induced phosphorylation has been extensively studied, the alternative Ser389 phosphorylation by p38 MAPK remains poorly characterized. This inhibitory phosphorylation specific for the GSK3β isoform, was originally described as a regulator of immunity and survival after DNA damage. Although phosphorylation of GSK3β at Ser389 is selectively abundant in the brain under physiological conditions, the function of this mechanism of GSK3β regulation in the brain, including development and neuroimmunity, is unknown. Here we examine in mouse: 1) the developmental regulation and regional distribution of brain phospho-Ser389 GSK3β expression, in comparison to classical Akt-induced phosphorylation; 2) neural cell types that express Ser389 GSK3β specificity and its regulation by genotoxic damage, and 3) the implication of GSK3β Ser389 phosphorylation in the neuroimmune response to lipopolysaccharide; using GSK3β Ser389 Ala knock-in mice, and applying a method of brain cell isolation that we previously optimized for flow cytometry evaluation. We found evidence that phospho-Ser389 is regional and developmentally regulated in the brain, with significantly higher expression of phospho-Ser389 GSK3β in neurons than in microglia, astrocytes and neural precursor cells. However, genotoxic damage increases nuclear phospho-Ser389 GSK3β expression in all neural cell types examined. We also present a comparative analysis of dissociation and Percoll-based separation methods for the combined isolation of healthy microglia, astrocytes and infiltrated lymphocytes suitable for flow cytometry, distinguishing between neonatal and adult mouse brain. The application of this optimized procedure and GSK3β knock-in mice led us to uncover the key role of GSK3β Ser389 phosphorylation in both, glial cell activation and immune cell infiltration in the brain; while effects observed on peripheral immunity were limited. Taken together, the results obtained in this thesis confirm that GSK3β Ser389 phosphorylation is a crucial mechanism for GSK3β inactivation in the brain largely overlooked, with a potential implication in neurological diseases that urges further investigation.