Control of reactive astrocytes by Notch1 and Amyloid Precursor Protein

描述（由申请人提供）：反应性星形胶质增生和随后形成的胶质瘢痕是各种中枢神经系统损伤后发生的强大现象。令人惊讶的是，控制反应性星形胶质细胞增殖或其体内功能的分子信号知之甚少。明确反应性星形胶质细胞增殖和功能的分子控制可能会导致改变反应性星形胶质细胞中特定信号的治疗策略，以保护组织或改善中枢神经系统损伤后的恢复。我们证明脑卒中后动脉内输注伽马分泌酶（GS）抑制剂DBZ（二苯氮平）可显著降低脑梗死周围皮层反应性星形胶质细胞的增殖，显著降低胶质纤维酸性蛋白（GFAP，活化的肥大星形胶质细胞的标志）的表达，并显著增加脑卒中梗死体积。卒中和DBZ治疗后反应性星形胶质细胞的缺失与侵袭卒中半暗带的cd45阳性炎症细胞数量的显著增加相关。同样，与载体注射对照相比，直接向皮质立体定向注射DBZ减少了脑刺伤周围（针迹）增殖反应性星形胶质细胞的数量。注射DBZ后，脑刺伤周围的反应性星形胶质细胞形态发生改变，平均突起数量、分支点数量和分支末端数量显著减少。GS裂解产物特异性抗血清免疫组化显示皮质损伤后反应性星形胶质细胞中NICD1 （Notch1）和AICD的核定位。DBZ阻断了GS的一种成分——早老素1的催化活性。在脑卒中前使用条件敲除小鼠特异性地从反应性星形胶质细胞中删除Notch1和APP的实验表明，两者都能调节脑卒中后梗死周围区域的皮质反应性星形胶质细胞。我们的研究结果表明，早老素1、Notch1和APP调节脑卒中后的星形胶质细胞反应性。具体目标：1；确定早老素1是否作为脑卒中后反应性星形胶质细胞形成的全局调节剂。2. 确定Notch1或APP信号是否控制脑卒中后反应性星形胶质细胞的增殖、形态和/或抗炎功能。3. 确定反应性星形胶质细胞中早老素1、Notch1或APP的表达对脑卒中后血脑屏障的修复是否必要。

DESCRIPTION (provided by applicant): Reactive astrogliosis and the subsequent formation of a glial scar are robust phenomena that occur following diverse CNS injuries. Surprisingly, the molecular signals that control the proliferation of reactive astrocytes or their functions in vivo are poorly understood. Defining the molecular control of reactive astrocyte proliferation and function may lead to therapeutic strategies that modify specific signals in reactive astrocytes to preserve tissue or improve recovery after CNS injury. We demonstrate that intra-arterial infusion of the gamma secretase (GS) inhibitor DBZ (Dibenzazepine) after stroke significantly reduced the proliferation of reactive astrocytes in the peri-infarct area of the cortex, significantly reduced the expression of glial fibrillary acidic protein (GFAP, a marker of activated hypertrophic astrocytes), and significantly increased stroke infarct volumes. The absence of reactive astrocytes after stroke and DBZ treatment correlated with a significant increase in the number of CD45-positive inflammatory cells that invaded the stroke penumbra. Similarly, stereotaxic injection of DBZ directly into the cortex reduced the numbers of proliferating reactive astrocytes surrounding the brain stab injury (needle track) compared with vehicle-injected controls. Reactive astrocytes surrounding the brain stab injury that remained after DBZ injection possessed an altered morphology with a significant reduction in average number of processes, number of branch points, and number of branch ends. Immunohistochemistry with antisera specific to GS cleavage products demonstrated nuclear localization of NICD1 (Notch1) and AICD in reactive astrocytes after cortical injury. DBZ blocks the catalytic activity of Presenilin 1, a component of GS. Experiments designed to specifically delete Notch1 and APP from reactive astrocytes prior to stroke using conditional knockout mice demonstrated that both regulate cortical reactive astrocytes in the peri-infarct area after stroke. Collectively our results indicate that Presenilin 1, Notch1, and APP regulate reactive astrocytes after stroke. Specific Aims: 1. To determine if Presenilin 1 acts as a global regulator of reactive astrogliosis after stroke. 2. To determine whether Notch1 or APP signaling controls the proliferation, morphology, and/or anti-inflammatory functions of reactive astrocytes after stroke. 3. To determine whether Presenilin 1, Notch1, or APP expression in reactive astrocytes is necessary for repair of the blood brain barrier after stroke.