Glycogen synthase kinase-3 regulates microglial migration, inflammation, and inflammation-induced neurotoxicity
Introduction
Microglia, the resident immune cells of the brain, constantly survey their microenvironment through process extension and retraction in order to respond to homeostatic disturbances, such as injury, disease, or infection [1], [2]. Resting microglia exhibit a ramified morphology, but upon stimulation, retract their processes and undergo directed chemotaxis along gradients of molecules released by injured cells (e.g., extracellular ATP) or by other immune cells (e.g., CCL2/MCP-1). A commonly used marker of microglial activation is the upregulation of the constitutively expressed CD11b, the αMβ2 integrin and complement receptor 3 [3]. Activated microglia produce an array of inflammatory molecules, act as antigen presenting cells, and phagocytose damaged cells [4]. One of the major products of activated microglia is the proinflammatory cytokine interleukin-6 (IL-6). Exposure to lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, activates microglia to produce IL-6 as well as other inflammatory molecules, such as nitric oxide (NO). The inflammatory responses of microglia are tightly regulated, but it is thought that excessive or chronic microglial activation can contribute to neurodegenerative processes [5]. Therefore, modulation of microglia responses may provide a therapeutic target for the treatment of severe or chronic neuroinflammatory conditions.
We recently reported [6] that LPS-stimulated production of IL-6 by monocytes was attenuated by inhibitors of glycogen synthase kinase-3 (GSK3), a constitutively active serine–threonine kinase [7]. Therefore, in order to investigate if neuroinflammation is regulated by GSK3, in the present study we tested if GSK3 inhibitors are able to attenuate LPS-stimulated proinflammatory cytokines produced by microglia as well as regulating the induction of iNOS and cyclooxygenase-2 (COX-2). GSK3 also regulates the motility of a number of types of cells [8]. Migration is a critical component of the microglial response to inflammatory stimuli, yet no studies have examined if GSK3 regulates microglial migration. Therefore, we tested if GSK3 regulates microglial migration both in vitro and in situ in acute hippocampal slices. Altogether, the results show that GSK3 inhibitors reduce microglial migration and attenuate the production of inflammatory molecules by activated microglia. Importantly, the results also demonstrate that the attenuation of microglial activity by GSK3 inhibitors provides neuroprotection during neuroinflammatory conditions, indicating that GSK3 is a potential therapeutic target to attenuate neuroinflammation.
Section snippets
Reagents and cells
Reagents were obtained from the following sources: LiCl (Sigma, St. Louis, MO), kenpaullone, indirubin-3′-monoxime (Alexis Biochemicals, San Diego, CA), CHIR99021 (University of Dundee), SB216763 and SB415286 (Tocris, Ellisville, MO), CCL2 (R&D Systems, Minneapolis, MN), SB203580, D4476, and roscovitine (Calbiochem, La Jolla, CA). Protein-free E. coli (K235) LPS was a generous gift from Dr. S. Michalek, and was prepared as previously described [9]. Mouse microglia BV-2 cells (a gift from Dr. E.
GSK3 regulates both random and directed migration of microglia in vitro
We tested if GSK3 regulates BV-2 microglial migration using two methods, a scratch assay and a transwell migration assay. During a 6 h incubation period after implementation of a scratch, BV-2 microglia migrated into the scratched zone sufficiently to reduce the depleted area by 53% (Fig. 1A). To assess the effect of GSK3 on microglial migration, cells were incubated with three structurally distinct selective GSK3 inhibitors, lithium [16], indirubin-3′-monoxime [17], or kenpaullone [18], for
Discussion
The results of this study reveal that GSK3 is an important regulator of microglia. GSK3 promoted both microglial migration and the production of inflammatory molecules by microglia, and also promoted inflammation-induced neurotoxicity. Specifically, microglial migration assessed either with cultured microglia in two experimental paradigms or in acute hippocampal slices was reduced by GSK3 inhibitors. Moreover, in LPS-stimulated microglia, inhibitors of GSK3 greatly reduced the production of
Conclusions
Microglia play an important role in neuroinflammatory conditions. Initial inflammatory responses appear to be beneficial for contributing to the resolution of the inflammation-inducing insult, but chronic inflammation appears be deleterious and to contribute to the progression of neurodegenerative diseases [5]. Understanding the intracellular signaling processes that regulate microglial responses is critical for developing novel therapeutics for neuroinflammatory conditions. The present study
Acknowledgments
The authors are grateful to Dr. S. Michalek for providing the purified LPS, Dr. E. Benveniste for providing the BV-2 cells, Dr. E. Beurel for critical discussions, Dr. H. Sontheimer for use of his microscope facilities and assistance, Anna Zmijewska and Gordon Meares for experimental assistance, and Dr. K. Roth and the UAB Neuroscience Core Facilities (NS47466, NS57098). This research was supported by a Civitan Emerging Scholars award and grants from the National Institutes of Health (MH38752,
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