Phosphoinositide 3-Kinase γ Restrains Neurotoxic Effects of Microglia After Focal Brain Ischemia
- 357 Downloads
Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and phagocytosis. This study was conducted to elucidate conjectural differences of lipid kinase-dependent and kinase-independent functions of PI3Kγ in the evolvement of brain damage induced by focal cerebral ischemia/reperfusion. Therefore, PI3Kγ wild-type, knockout, and kinase-dead mice were subjected to middle cerebral artery occlusion followed by reperfusion. Tissue damage and cellular composition were assessed by immunohistochemical stainings. In addition, microglial cells derived from respective mouse genotypes were used for analysis of PI3Kγ effects on phagocytic activity, matrix metalloproteinase-9 release, and cAMP content under conditions of oxygen/glucose deprivation and recovery. Brain infarction was more pronounced in PI3Kγ-knockout mice compared to wild-type and kinase-dead mice 48 h after reperfusion. Immunohistochemical analyses revealed a reduced amount of galectin-3/MAC-2-positive microglial cells indicating that activated phagocytosis was reduced in ischemic brains of knockout mice. Cell culture studies disclosed enhanced metalloproteinase-9 secretion in supernatants derived from microglia of PI3Kγ-deficient mice after 2-h oxygen/glucose deprivation and 48-h recovery. Furthermore, PI3Kγ-deficient microglial cells showed a failed phagocytic activation throughout the observed recovery period. Lastly, PI3Kγ-deficient microglia exhibited strongly increased cAMP levels in comparison with wild-type microglia or cells expressing kinase-dead PI3Kγ after oxygen/glucose deprivation and recovery. Our data suggest PI3Kγ kinase activity-independent control of cAMP phosphodiesterase as a crucial mediator of microglial cAMP regulation, MMP-9 expression, and phagocytic activity following focal brain ischemia/recirculation. The suppressive effect of PI3Kγ on cAMP levels appears critical for the restriction of ischemia-induced immune cell functions and in turn tissue damage.
KeywordsMCAO PI3Kγ Microglial cells Neuroinflammation
The authors acknowledge Mrs. M. Guenther, Mrs. S. Tausch and Mrs. R.-M., Zimmer for skillful technical assistance, and F. D. Boehmer, for his collegial editorial review of the manuscript. The study was supported by the Deutsche Forschungsgemeinschaft (Grant RTG 1715) and by the German Federal Ministry of Education and Research (BMBF; Grant FKZ 01EO1002; Center for Sepsis Control and Care). C.S and N.S. are PhD students of the Research Training Group 1715 “Adaptive Stress Responses” Grant RTG 1715 of the DFG, and C.S. was supported in part by the Integrated Research and Treatment Center, Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany.
C.S. designed and carried out the in vitro studies and analyzed the data. C.F. designed and supervised the animal experiments. N.S. carried out in vitro studies and analyzed the data. J.P.M. supervised the in vitro studies and revised the manuscript. M.B. carried out the immunohistochemical studies and analyzed the data. I.F. carried out the chimeric experiments. O.W.W. contributed to the study design, supervised the animal experiments, and revised the manuscript. E.H. designed and supervised the chimeric experiments. R.W. designed and coordinated the study and revised the manuscript. R.B. designed the study, supervised the experiments, analyzed the data, and wrote the manuscript.
Compliance with Ethical Standards
Conflicts of Interest
Nothing to report.
- 16.Murga C, Laguinge L, Wetzker R, Cuadrado A, Gutkind JS (1998) Activation of Akt/protein kinase B by G protein-coupled receptors. A role for alpha and beta gamma subunits of heterotrimeric G proteins acting through phosphatidylinositol-3-OH kinasegamma. J Biol Chem 273(30):19080–19085PubMedCrossRefGoogle Scholar
- 18.Stoyanov B, Volinia S, Hanck T, Rubio I, Loubtchenkov M, Malek D, Stoyanova S, Vanhaesebroeck B, Dhand R, Nurnberg B, Gierschik P, Seedorf K, Hsuan JJ, Waterfield MD, Wetzker R (1995) Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science 269(5224):690–693PubMedCrossRefGoogle Scholar
- 20.Patrucco E, Notte A, Barberis L, Selvetella G, Maffei A, Brancaccio M, Marengo S, Russo G, Azzolino O, Rybalkin SD, Silengo L, Altruda F, Wetzker R, Wymann MP, Lembo G, Hirsch E (2004) PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent and -independent effects. Cell 118(3):375–387PubMedCrossRefGoogle Scholar
- 21.Frister A, Schmidt C, Schneble N, Brodhun M, Gonnert FA, Bauer M, Hirsch E, Muller JP, Wetzker R, Bauer R (2014) Phosphoinositide 3-Kinase gamma Affects LPS-Induced Disturbance of Blood–brain Barrier Via Lipid Kinase-Independent Control of cAMP in Microglial Cells. Neuromol Med 16(4):704–713CrossRefGoogle Scholar
- 34.Bianco F, Fumagalli M, Pravettoni E, D'Ambrosi N, Volonte C, Matteoli M, Abbracchio MP, Verderio C (2005) Pathophysiological roles of extracellular nucleotides in glial cells: differential expression of purinergic receptors in resting and activated microglia. Brain Res Brain Res Rev 48(2):144–156PubMedCrossRefGoogle Scholar
- 36.Rivera S, Ogier C, Jourquin J, Timsit S, Szklarczyk AW, Miller K, Gearing AJ, Kaczmarek L, Khrestchatisky M (2002) Gelatinase B and TIMP-1 are regulated in a cell- and time-dependent manner in association with neuronal death and glial reactivity after global forebrain ischemia. Eur J Neurosci 15(1):19–32PubMedCrossRefGoogle Scholar
- 41.Stoll G, Schroeter M, Jander S, Siebert H, Wollrath A, Kleinschnitz C, Bruck W (2004) Lesion-associated expression of transforming growth factor-beta-2 in the rat nervous system: evidence for down-regulating the phagocytic activity of microglia and macrophages. Brain Pathol 14(1):51–58PubMedCrossRefGoogle Scholar
- 42.Schilling M, Besselmann M, Muller M, Strecker JK, Ringelstein EB, Kiefer R (2005) Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice. Exp Neurol 196(2):290–297PubMedCrossRefGoogle Scholar
- 43.Perino A, Ghigo A, Ferrero E, Morello F, Santulli G, Baillie GS, Damilano F, Dunlop AJ, Pawson C, Walser R, Levi R, Altruda F, Silengo L, Langeberg LK, Neubauer G, Heymans S, Lembo G, Wymann MP, Wetzker R, Houslay MD, Iaccarino G, Scott JD, Hirsch E (2011) Integrating cardiac PIP3 and cAMP signaling through a PKA anchoring function of p110gamma. Mol Cell 42(1):84–95PubMedPubMedCentralCrossRefGoogle Scholar