No abstract

The inhibition of Glycogen Synthase Kinase 3 β (GSK3β) by Ser9 phosphorylation affects many physiological processes, including the immune response. However, the consequences of GSK3β inhibition by alternative Ser389 phosphorylation remain poorly characterized. Here we have examined neuroinflammation in GSK3β Ser389 knock-in (KI) mice, in which the phosphorylation of Ser389 GSK3β is impaired. The number of activated microglia/infiltrated macrophages, astrocytes, and infiltrated neutrophils was significantly higher in these animals compared to C57BL/6J wild-type (WT) counterparts, which suggests that the failure to inactivate GSK3β by Ser389 phosphorylation results in sustained low-grade neuroinflammation. Moreover, glial cell activation and brain infiltration of immune cells in response to lipopolysaccharide (LPS) failed in GSK3β Ser389 KI mice. Such effects were brain-specific, as peripheral immunity was not similarly affected. Additionally, phosphorylation of the IkB kinase complex (IKK) in response to LPS failed in GSK3β Ser389 KI mice, while STAT3 phosphorylation was fully conserved, suggesting that the NF-κB signaling pathway is specifically affected by this GSK3β regulatory pathway. Overall, our findings indicate that GSK3β inactivation by Ser389 phosphorylation controls the brain inflammatory response, raising the need to evaluate its role in the progression of neuroinflammatory pathologies.

Endo-lysosomes are considered acidic Ca2+ stores but direct measurements of luminal Ca2+ within them are limited. Here we report that the Ca2+-sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca2+ dynamics in this compartment. We show that Ca2+ uptake is ATP-dependent and sensitive to blockers of endoplasmic reticulum Ca2+ pumps. We find that the Ca2+ mobilizing messenger IP3 which typically targets the endoplasmic reticulum evokes robust luminal responses in wild type cells, but not in IP3 receptor knock-out cells. Responses were comparable to those evoked by activation of the endo-lysosomal ion channel TRPML1. Stimulation with IP3-forming agonists also mobilized the store in intact cells. Our data reveal a physiologically-relevant, IP3-sensitive store of Ca2+ within the endo-lysosomal system.

GSK3β is a constitutively active kinase that promotes cell death, which requires strict regulatory mechanisms. Although Aktmediated phosphorylation at Ser 9 is the default mechanism to inactivate GSK3β, phosphorylation of GSK3β at Ser 389 by p38 MAPK has emerged as an alternative inhibitory pathway that provides cell protection and repair in response to DNA damage. Phosphorylation of Ser 389 GSK3β has been detected in adult brain, where it has been related to neuronal survival and behavior. However, the use of this pathway to regulate GSK3β in the neonatal developing brain is unknown. In this study, we show that phosphorylation of GSK3β at Ser 389 in the brain is developmentally regulated, with the highest levels corresponding to the first 2 weeks of age. Moreover, we found that the phosphorylation of GSK3β at Ser 389 is the preferential mechanism for inactivating brain GSK3β in 2-week-old mice. Importantly, we show that phospho-Ser 389 GSK3β expression is predominant in neuronal cell cultures from neonatal brain relative to other cell populations. However, phospho-Ser 389 GSK3β is triggered by DNA doublestrand breaks in all developing neural cell types examined. Thus, the phosphorylation of GSK3β on Ser 389 could be a central regulatory mechanism to restrain GSK3β during neurogenesis early in life. Keywords p38 MAPK . DNA double-strand breaks (DSBs) . Astrocytes . Microglia . Neurons . Neural precursor cells (NPCs)