P.1.a.020 Pharmacogenetic response to antidepressant and suicidality in a large cohort of outpatients with a major depression episode

Abstract

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP2 is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP2 in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP2 increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP2. In contrast, PIP5Kα knockdown that reduced PIP2 levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP2-dependent recruitment of TIRAP to the plasma membrane.Background: Phosphoinositides are involved in regulating TLR4 signaling.Results: PIP5Kα knockdown in BV2 microglial cells inhibits LPS-induced inflammatory responses, PIP2 increase, and TIRAP translocation to the plasma membrane.Conclusion: PIP5Kα-derived PIP2 facilitates TLR4-mediated microglial inflammatory responses through recruitment of TIRAP to the plasma membrane.Significance: Regulation of PIP5Kα-dependent PIP2 pool may modulate TLR4-associated immune function in microglia.