ABSTRACT
Vitamin C is essential for the development and function of the central nervous system (CNS). The plasma membrane sodium-vitamin C cotransporter 2 (SVCT2) is the primary mediator of vitamin C uptake in neurons. SVCT2 specifically transports ascorbate, the reduced form of vitamin C, which acts as a reducing agent. We demonstrated that ascorbate uptake through SVCT2 was critical for the homeostasis of microglia, the resident myeloid cells of the CNS that are essential for proper functioning of the nervous tissue. We found that depletion of SVCT2 from the plasma membrane triggered a proinflammatory phenotype in microglia and resulted in microglia activation. Src-mediated phosphorylation of caveolin-1 on Tyr14 in microglia induced the internalization of SVCT2. Ascorbate treatment, SVCT2 overexpression, or blocking SVCT2 internalization prevented the activation of microglia. Overall, our work demonstrates the importance of the ascorbate transport system for microglial homeostasis and hints that dysregulation of ascorbate transport might play a role in neurological disorders.
Subject(s)
Ascorbic Acid/metabolism , Caveolin 1/metabolism , Endocytosis , Microglia/metabolism , Neurons/metabolism , Sodium-Coupled Vitamin C Transporters/metabolism , Animals , Blotting, Western , Cell Line , Cell Membrane/metabolism , Cytokines/metabolism , Female , HEK293 Cells , Humans , Inflammation Mediators/metabolism , Male , Mice, Knockout , Microglia/cytology , Microscopy, Confocal , Phosphorylation , Rats, Wistar , Sodium-Coupled Vitamin C Transporters/geneticsABSTRACT
Microglial cells are the resident macrophages of the central nervous system. Their function is essential for neuronal tissue homeostasis. After inflammatory stimuli, microglial cells become activated changing from a resting and highly ramified cell shape to an amoeboid-like morphology. These morphological changes are associated with the release of proinflammatory cytokines and glutamate, as well as with high phagocytic activity. The acquisition of such phenotype has been associated with activation of cytoplasmic tyrosine kinases, including those of the Src family (SFKs). In this study, using both in vivo and in vitro inflammation models coupled to FRET-based time-lapse microscopy, lentiviruses-mediated shRNA delivery and genetic gain-of-function experiments, we demonstrate that among SFKs c-Src function is necessary and sufficient for triggering microglia proinflammatory signature, glutamate release, microglia-induced neuronal loss, and phagocytosis. c-Src inhibition in retinal neuroinflammation experimental paradigms consisting of intravitreal injection of LPS or ischemia-reperfusion injury significantly reduced microglia activation changing their morphology to a more resting phenotype and prevented neuronal apoptosis. Our data demonstrate an essential role for c-Src in microglial cell activation.
