Plasmalemma permeability and necroptosis: New targets for intracerebral hemo

描述（由申请人提供）：非外伤性脑出血（ICH）是一个缺乏特异性治疗的重大公共卫生问题。我们提出了几条证据表明，质膜通透性和坏死有助于胶原酶诱导小鼠脑出血后细胞死亡。脑出血后，质膜对碘化丙啶（PI）的通透性是细胞损伤和死亡的关键特征；TNF α和Fas受体缺乏小鼠脑出血后质膜通透性降低50%；坏死样细胞死亡，通过电镜、组织学和生化标准评估，发生在脑出血后；和PI+细胞在脑出血后通过敲除RIPK3（控制程序性坏死的关键分子）而减少。这些初步研究结果强烈表明，脑出血后坏死下垂（由TNF/Fas引发，由RIPK1和RIPK3介导）有助于细胞死亡，并可能导致神经功能障碍。坏死他汀类药物是RIPK1的小分子抑制剂，在脑外伤和缺血性中风模型中抑制细胞坏死，保护细胞死亡和功能缺陷。Kollidon VA64是FDA批准的药丸制造配方成分，可在体内重新密封CCI和ICH后受损细胞。给药VA64可减轻小鼠CCI后血脑屏障损伤、脑水肿、细胞死亡和运动功能障碍，并可使损伤细胞重封，减轻胶原酶ICH后血脑屏障损伤。利用小鼠胶原酶和自体血液脑出血模型，我们提出了3个具体目标来验证中心假设：(1)质膜损伤是脑出血后细胞死亡的标志和中介，(2)程序性坏死有助于脑出血后细胞死亡和功能结局。目的1将使用脉冲标记实验来跟踪体内损伤细胞的命运，并验证质膜完整性丧失是脑出血后致死性细胞损伤的生物标志物的假设；目的2将验证VA64在脑出血后恢复质膜完整性、减少继发性损伤和挽救损伤脑细胞免于死亡的假设；目的3将通过RIPK3基因敲除了小鼠和坏死性他汀类药物，以及使用坏死性他汀类药物加VA64的翻译方法，验证坏死性上闭诱导脑出血后质膜完整性丧失和细胞死亡的假设。这些目标的完成将使坏死成为脑出血的核心，并可能使VA64和坏死他汀类药物成为脑出血的新疗法。

DESCRIPTION (provided by applicant): Non-traumatic intracerebral hemorrhage (ICH) is a major public health problem that lacks specific therapy. We present several lines of evidence suggesting that plasmalemma permeability and necrosis contribute to cell death after collagenase induced ICH in mice. Following ICH, plasmalemma permeability to propidium iodide (PI) is a key feature of cellular injury and death; plasmalemma permeability after ICH is reduced by 50% in mice deficient in TNF alpha and Fas receptor; necrotic-like cell death, assessed by EM, histological, and biochemical criteria occurs after ICH; and PI+ cells are reduced after ICH by RIPK3 knockout (a key molecule governing programmed necrosis). These preliminary findings strongly suggest that necroptosis (initiated by TNF/Fas and mediated by RIPK1 and RIPK3) contribute to cell death, and perhaps neurological dysfunction, after ICH. Necrostatins are small molecule inhibitors of RIPK1 that inhibit necroptosis in cells and protect against cell death and functional deficits in brain trauma and ischemic stroke models. Kollidon VA64 is a FDA approved formulary component in pill manufacture that reseals injured cells after CCI and ICH in vivo. VA64 administration reduces blood brain barrier damage, brain edema, cell death, and motor dysfunction after CCI, and reseals injured cells and reduces BBB damage after collagenase ICH in mice. Using mouse collagenase and autologous blood ICH models, we propose 3 specific aims to test the central hypotheses that (1) plasmalemma damage is a marker and mediator of cell death after ICH, and (2) that programmed necrosis contributes to cell death and functional outcome after ICH. Aim 1 will use pulse labeling experiments to follow the fate of injured cells in vivo and test the hypothesis that loss of plasmalemma integrity is a biomarker of fatal cellular injury after ICH; Aim 2 will test the hypothesis that VA64 restores plasmalemma integrity, reduces secondary injury and rescues injured brain cells from death after ICH; Aim 3 will test the hypothesis that necroptosis induces loss of plasmalemma integrity and cell death after ICH, using RIPK3 knockout mice and necrostatins, and a translational approach using necrostatins plus VA64. Completion of these Aims will establish necrosis as central to ICH, and may establish VA64 and necrostatins as novel therapies for ICH.