Control of reactive astrocytes by Notch1 and Amyloid Precursor Protein

描述(由申请人提供)：反应性星形胶质细胞增生症和随后形成的胶质瘢痕是发生在各种中枢神经系统损伤后的强烈现象。令人惊讶的是，控制反应性星形胶质细胞增殖的分子信号或它们在体内的功能尚不清楚。明确反应性星形胶质细胞增殖和功能的分子调控可能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，卒中后动脉内注入伽马分泌酶(GS)抑制剂DBZ(二苯扎西平)可显著减少大脑皮质梗死区反应性星形胶质细胞的增殖，显著减少激活的肥大星形胶质细胞的标志物--胶质纤维酸性蛋白(GFAP)的表达，并显著增加卒中梗死体积。卒中和DBZ治疗后反应性星形胶质细胞的缺失与侵入卒中半暗带的CD45阳性炎症细胞的数量显著增加有关。类似地，脑刺伤(针迹)周围脑刺伤(针迹)周围的星形胶质细胞增殖反应性星形胶质细胞数量与车辆注射对照相比减少。注射DBZ后残留在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、分支点数和分支末端数显著减少。用GS裂解产物的抗血清进行免疫组织化学染色，显示NICD1(Notch1)和AICD在皮质损伤后反应性星形胶质细胞中的核定位。DBZ阻断GS组分早老素1的催化活性。利用条件性基因敲除小鼠在中风前从反应性星形胶质细胞中专门删除Notch1和APP的实验表明，这两种药物都能调节中风后梗塞周围区域的皮质反应性星形胶质细胞。总而言之，我们的结果表明，早老素1、Notch1和APP调节卒中后反应性星形胶质细胞。具体目的：1.确定早老素1是否在卒中后反应性星形胶质细胞增生症中起整体调节作用。2.确定Notch1或APP信号通路是否控制卒中后反应性星形胶质细胞的增殖、形态和/或抗炎功能。3.探讨卒中后血脑屏障修复是否需要反应性星形胶质细胞表达早老素1、Notch1或APP。

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.