Elsevier

Cellular Signalling

Volume 21, Issue 2, February 2009, Pages 264-273
Cellular Signalling

Glycogen synthase kinase-3 regulates microglial migration, inflammation, and inflammation-induced neurotoxicity

https://doi.org/10.1016/j.cellsig.2008.10.014Get rights and content

Abstract

Microglia play a prominent role in the brain's inflammatory response to injury or infection by migrating to affected locations, secreting inflammatory molecules, and phagocytosing damaged tissue. However, because severe or chronic neuroinflammation exacerbates many neurological conditions, controlling microglia actions may provide therapeutic benefits in a diverse array of diseases. Since glycogen synthase kinase-3 (GSK3) promotes inflammatory responses in peripheral immune cells, we investigated if inhibitors of GSK3 attenuated microglia responses to inflammatory stimuli. Treatment of BV-2 microglia with GSK3 inhibitors greatly reduced the migration of microglia in both a scratch assay and in a transwell migration assay. Treatment of BV-2 microglia with lipopolysaccharide (LPS) stimulated the production of interleukin-6 and increased the expression of inducible nitric oxide synthase (iNOS) and NO production. Each of these microglia responses to inflammatory stimulation were greatly attenuated by GSK3 inhibitors. However, GSK3 inhibitors did not cause a general impairment of microglia functions, as the LPS-induced stimulated expression of cylcooxygenase-2 was unaltered. Regulation of microglia functions were also evident in cultured mouse hippocampal slices where GSK3 inhibitors reduced cytokine production and microglial migration, and provided protection from inflammation-induced neuronal toxicity. These findings demonstrate that GSK3 promotes microglial responses to inflammation and that the utilization of GSK3 inhibitors provides a means to limit the inflammatory actions of microglia.

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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