Abstract
Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca2+]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion.
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich TRR 43 and Cluster of Excellence 257 NeuroCure), VolkswagenStiftung (Lichtenberg Program to M. E.), the Bundesministerium für Bildung und Forschung (Center for Stroke Research Berlin) and the European Union’s Seventh Framework Program (Grant No. FP7/2008–2013) under grant Agreement Nos. 201024 and 202213 (European Stroke Network). The authors wish to thank Bettina Herrmann, Susann Eigel and Stefanie Balz for excellent technical assistance.
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M. Endres and G. Kronenberg contributed equally as last authors.
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Supplementary Fig. 1 Isolation and in vitro cultivation of adult microglia. The procedure for the cultivation of adult-derived microglia has been described in detail previously (Scheffel et al. 2012). a Astrocytic “substrate” cultures were derived from neonatal wild-type mice. b Brain lysates from Gsn +/+ and Gsn −/− brains were added to the substrate cultures. c The purity of these microglial cultures was confirmed by genotyping PCR (PDF 32 kb)
429_2015_1067_MOESM2_ESM.pdf
Supplementary Fig. 2 Microglia density in Gsn +/+ and Gsn −/− mouse brain. The density of Iba1+ cells was quantified in hippocampus (a) and striatum (b) of adult (<6 months) and aged (>16 months) Gsn +/+ and Gsn −/− mice. *p<0.05, two-way ANOVA followed by Tukey’s multiple comparisons test. N = 6 mice per group; Scale bars 100 µM (PDF 204 kb)
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Uhlemann, R., Gertz, K., Boehmerle, W. et al. Actin dynamics shape microglia effector functions. Brain Struct Funct 221, 2717–2734 (2016). https://doi.org/10.1007/s00429-015-1067-y
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DOI: https://doi.org/10.1007/s00429-015-1067-y