Protective role of Neuregulin-1 against cerebral malaria-induced neuronal injury and behavioral sequelae

人脑疟疾(HCM)是恶性疟原虫(P.F.)的一种严重类型。与疟疾相关的 每年约有50万儿童死亡，一些幸存者的大脑功能受损。HCM的特点是 寄生红细胞在脑微循环中的滞留及其致炎作用 中介物、脑肿胀和意识受损并伴有无法唤醒的昏迷。我们已经确定 循环中的游离血红素和pRBC裂解的副产物寄生虫富含组氨酸蛋白2(HRP2)是主要原因 与肥厚性脑炎相关的脑部炎症、血脑屏障(BBB)功能障碍和脑损伤。然而， 在缺乏合适的模型的情况下，定义在HCM中调节这些效应的机制(S)是具有挑战性的。在……里面 体外2D细胞培养、3D脑器官和动物模型(ECM；C57BL/6中的伯氏疟原虫Anka) 提示血红素和HRP2诱导细胞凋亡、炎症和组织紊乱。在ECM中， 血红素引起脑血管内皮细胞凋亡，改变血管生成素1和2的比例，上调CXCL10， 血红素加氧酶1和tau以及通过基质中的STAT3信号影响血脑屏障的完整性 金属蛋白酶3(MMP3)。在筛选抗ECM的治疗剂后，我们发现 NeuRegin-1(NRG1)是一种8 kDa的神经肽，目前正在进行治疗心力衰竭的临床试验， 以5微克/公斤的速度静脉注射可减弱细胞外基质。NRG1介导ErbB4(受体)的磷酸化， 激活人脑微血管内皮细胞中的AKT和失活STAT3。ECM耐药小鼠 BALB/c小鼠脑组织中NRG1的表达水平高于ECM易感小鼠(C57BL/6)。 由于致命性HCM和ECM循环中的NRG1都严重耗尽，因此需要评估NRG1的水平 前瞻性地确定加强治疗以减轻HCM严重程度所需的量。有趣的是，CD8 T细胞 PD1/PD-L1信号转导细胞外基质恢复，PD1被NRG1上调。使用人类干细胞- 衍生神经血管单位(NVU；脑芯片)、细胞外基质和人体，我们将通过以下方式确定其机制 NRG1可以减轻脑型疟疾。我们假设NRG1的治疗性给药会减弱 血红素和HRP2通过NRG1/ErbB4和PD/PD-L1信号诱导NVU损伤和ECM死亡。我们的 目的是从功能上评估NRG1介导的抑制ECM的关键调控通路，以及血红素- HRP2诱导的NVU损伤。其具体目标是：1)测试算法的假设，包括 NRG1、血红素、HRP2以及神经元损伤和炎症的标志物可以预测HCM的严重程度和死亡率 和神经行为后遗症；2)检验假设NRG1/ErbB4和PD1/PD-L1信号串扰 保护血红素和HRP2诱导的人类NVU的损伤；以及3)检验NRG1的假设 通过NRG1/ErbB4和PD1/PD-L1信号通路减轻ECM脑损伤和行为缺陷。理解 NRG1在脑疟疾发病机制和幸存者后遗症中的作用将使我们能够确定 在临床试验中以NRG1为靶点的可行性，最终目的是改善肥厚型心肌炎儿童的存活率。

Human cerebral malaria (HCM) is a severe form of Plasmodium falciparum (P.f.) malaria associated with ~500,000 deaths in children annually and impaired brain function in some survivors. HCM is characterized by sequestration of parasitized red blood cells (pRBCs) in cerebral micro-circulation and induction of inflammatory mediators, brain swelling and impaired consciousness with unarousable coma. We have determined that circulating free heme and parasite histidine rich protein 2 (HRP2), by-products of pRBC lysis, are major causes of brain inflammation, blood-brain barrier (BBB) dysfunction, and brain injury associated with HCM. However, defining the mechanism(s) mediating these effects in HCM is challenging in the absence of suitable models. In vitro 2D cell culture, 3D brain organoid and animal models (ECM; Plasmodium berghei ANKA in C57BL/6) all indicate that heme and HRP2 induced cellular apoptosis, inflammation, and tissue disorganization. In ECM, heme causes brain vascular endothelial cell apoptosis, alters Angiopoietins 1 and 2 ratios, upregulates CXCL10, Heme Oxygenase 1, and tau as well as compromise BBB integrity through STAT3 signaling via matrix metalloproteinase three (MMP3). Following a screen for therapeutic agents against ECM, we identified Neuregulin- 1(NRG1), an 8 kDa neuropeptide currently undergoing clinical trials against heart failure, that attenuates ECM when delivered intravenously at 5µg/kg. NRG1 mediates phosphorylation of ErbB4 (receptor), activates AKT and inactivates STAT3 in human brain microvascular endothelial cells. ECM resistant mice (BALB/c) constitutively expressed higher levels of NRG1 in brain tissue than ECM susceptible (C57BL/6) mice. Since circulating NRG1 is severely depleted in both fatal HCM and ECM, levels of NRG1 need to be assessed prospectively to ascertain amounts needed for augmentation to mitigate HCM severity. Interestingly, CD8+Tcell PD1/PD-L1 signaling mediated ECM recovery and PD1 was upregulated by NRG1. Using a human stem cell- derived neurovascular unit (NVU; brain chip), ECM and human subjects, we will determine the mechanism by which NRG1 attenuates cerebral malaria. We hypothesize that therapeutic administration of NRG1 will attenuate heme and HRP2-induced NVU damage and ECM mortality via NRG1/ErbB4 and PD/PD-L1 signaling. Our objective is to functionally assess the key regulatory pathways mediated by NRG1 to attenuate ECM, and heme- and HRP2-induced NVU damage. The specific aims are: 1) to test the hypothesis that an algorithm, consisting of NRG1, heme, HRP2, and markers of neuronal injury and inflammation can predict HCM severity, mortality and neurobehavioral sequelae; 2) to test the hypothesis that NRG1/ErbB4 and PD1/PD-L1 signaling crosstalk protects against heme and HRP2-induced damage in human NVU; and 3) to test the hypothesis that NRG1 attenuates ECM brain injury and behavioral deficit via NRG1/ErbB4 and PD1/PD-L1 signaling. Understanding the role of NRG1 in cerebral malaria pathogenesis and sequelae in survivors will enable us to determine the feasibility of targeting NRG1 in clinical trials with the ultimate goal of improving the survival of children with HCM.