Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Friedrich Schiller University, Hans-Knöll-Strasse 2, D-07745, Jena, Germany.
Department of Neurology, Jena University Hospital, Jena, Germany.
Mol Neurobiol. 2016 Oct;53(8):5468-79. doi: 10.1007/s12035-015-9472-z. Epub 2015 Oct 9.
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.
磷酸肌醇 3-激酶 γ（PI3Kγ）与神经炎症和吞噬作用有关。本研究旨在阐明在局灶性脑缺血/再灌注引起的脑损伤演变过程中，PI3Kγ 的脂质激酶依赖性和非激酶依赖性功能的假设差异。因此，将 PI3Kγ 野生型、敲除型和激酶失活型小鼠进行大脑中动脉闭塞再灌注。通过免疫组织化学染色评估组织损伤和细胞组成。此外，还分析了来自不同小鼠基因型的小胶质细胞在氧/葡萄糖剥夺和恢复条件下对吞噬活性、基质金属蛋白酶-9 释放和 cAMP 含量的影响。与野生型和激酶失活型小鼠相比，PI3Kγ 敲除型小鼠在再灌注 48 小时后脑梗死更为明显。免疫组织化学分析显示，Galectin-3/MAC-2 阳性小胶质细胞数量减少，表明缺血性脑损伤中激活的吞噬作用减少。细胞培养研究显示，PI3Kγ 缺失型小鼠的小胶质细胞在 2 小时氧/葡萄糖剥夺和 48 小时恢复后，上清液中的金属蛋白酶-9 分泌增加。此外，PI3Kγ 缺失型小胶质细胞在整个观察到的恢复期间表现出吞噬作用激活失败。最后，与野生型小胶质细胞或表达激酶失活型 PI3Kγ 的细胞相比，PI3Kγ 缺失型小胶质细胞在氧/葡萄糖剥夺和恢复后 cAMP 水平显著升高。我们的数据表明，PI3Kγ 激酶活性非依赖性控制 cAMP 磷酸二酯酶作为小胶质细胞 cAMP 调节、MMP-9 表达和吞噬作用的关键介质，在局灶性脑缺血/再灌注后起作用。PI3Kγ 对 cAMP 水平的抑制作用对于限制缺血诱导的免疫细胞功能进而组织损伤至关重要。
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