Targeting induction of calcium buffer proteins for treatment of viral encephaliti

描述（由申请人提供）：病毒性脑炎治疗的一个关键障碍是在患者向医生报告病毒可能感染神经细胞后有效治疗的能力。不幸的是，神经元通常不具有神经再生能力，因此治疗方式应侧重于防止神经元死亡，以及用能够有效穿透血脑屏障的化合物限制病毒复制。本项目将制定一项战略来实现这一目标。该项目R21阶段的目的是确定增加钙缓冲蛋白是否可以明确保护培养物和动物中的神经细胞免受西尼罗病毒（WNV）诱导的死亡，并确定特定细胞代谢物在激活神经细胞中的钙缓冲蛋白方面的效用。R33期的目的是基于细胞培养功效和血脑屏障通透性鉴定铅代谢物，确定铅化合物在啮齿动物中的活性，并探讨这种治疗乙型脑炎病毒（JEV）的方法。具体目的是基于已发表的结果，即不同细胞系中的西尼罗河病毒感染导致ca2 +内流，这有利于其复制，但诱导caspase 3切割并增加细胞死亡的风险。相反，Ca++内流抑制剂可降低病毒滴度，减少caspase 3的切割，降低病毒滴度。在一种新型的神经系统疾病仓鼠模型中，calbindin D28k的诱导与神经元存活相关。相反，低基础水平的calbindin D28k与TUNEL染色增加和细胞损伤或死亡相关。此外，被研究的特定代谢物被公认为与受体共同转运到细胞核以激活钙缓冲蛋白的产生。具体目标是：R21 SA #1。验证钙缓冲蛋白可以保护培养物和动物的神经细胞免受西尼罗河病毒引起的死亡的假设。R21 sa #2。建立神经保护细胞培养实验，评估维生素D3代谢物对钙缓冲蛋白的诱导作用，并监测感染西尼罗河病毒的神经细胞的存活。R33 sa #1。使用神经保护细胞培养试验和跨内皮渗透性试验确定维生素D3代谢物的主要候选物。R33 sa #2。获得临床前数据，支持在不同给药途径和治疗方案的动物中使用候选先导治疗西尼罗河病毒脑炎。R33 sa #3。确定乙脑病毒的广谱治疗活性。由于在神经病毒学、抗病毒研究、维生素D代谢和钙信号方面的专业知识，我们在发现病毒性脑炎的治疗方面处于独特的地位。本项目可为西尼罗河病毒和乙脑病毒神经发病机制的基础研究提供新的途径，并为西尼罗河病毒、乙脑病毒和病毒性脑炎的治疗提供临床依据。

DESCRIPTION (provided by applicant): A critical barrier for the treatment of viral encephalitis is the ability to effectively treat patients after they present to their physicians when the virus as likely infected neuronal cells. Unfortunately, neurons are typically not neuro-regenerative, so therapeutic modalities should focus on preventing the death of neurons, as well as to limit virus replication with compounds that can effectively penetrate the blood brain barrier. This project will develop a strategy to accomplish this. The purpose of the R21 phase of the project is to determine if increased calcium buffer proteins can definitively protect neuronal cells in culture and animals from West Nile virus (WNV)-induced death, and to establish the utility of specific cellular metabolites to activate calcium buffer proteins in neuronal cells. The purpose of the R33 phase is to identify the lead metabolite based on cell culture-efficacy and blood brain barrier permeability, to establish activity of the lead compound in rodents, and to investigate this therapeutic approach with Japanese encephalitis virus (JEV). The specific aims are based on published results that WNV infection in different cell lines leads to Ca++ influx, which benefits is replication, but induces caspase 3 cleavage and increase the risk of cell death. Conversely, inhibitors of Ca++ influx decreases viral titer by >2 log10, and decreased caspase 3 cleavage. In an innovative hamster model of WN neurological disease, induction of calbindin D28k correlates with neuronal survival. Conversely, low basal levels of calbindin D28k correlates with increased TUNEL staining, and cell injury or death. Moreover, the specific metabolites to be investigated are well recognized to co-translocate to the nucleus with the receptor to activate production of calcium buffer proteins. The Specific Aims are: R21 SA #1. Test the hypothesis that calcium buffer proteins can protect neuronal cells in culture and animals from WNV-induced death. R21 SA #2. Establish a neuro- protection cell culture assay for evaluation of vitamin D3 metabolites for induction of calcium buffer proteins, and for monitoring the survival of WNV-infected neuronal cells. R33 SA #1. Identify lead candidate of vitamin D3 metabolites using the neuro-protective cell culture assay and a transendothelial permeability assay. R33 SA #2. Obtain preclinical data supporting the treatment of WNV encephalitis with lead candidate in animals with different routes of administration and treatment schedules. R33 SA #3. Determine the broad-spectrum therapeutic activity in JEV. Because of expertise in neurovirology, antiviral research, vitamin D metabolism, and calcium signaling, we are uniquely positioned to discover therapy for viral encephalitis. This project could provide new avenues of basic research for WNV and JEV neuro- pathogenesis, and enhance clinical practice for the management of WNV, JEV, and viral encephalitis in general. PUBLIC HEALTH RELEVANCE: The purpose of the R21 phase is to determine if increased calcium buffer proteins can definitively protect neuronal cells in culture and animals from West Nile virus (WNV)- induced death and to determine the utility of specific metabolites to activate calcium buffer proteins in neuronal cells. The purpose of the R33 phase is to identify the lead metabolite based on efficacy and blood brain barrier permeability, to establish activity of the lead compound in rodents for West Nile virus (WNV), and to investigate this therapeutic approach with Japanese encephalitis virus (JEV). To test the hypothesis that calcium buffer proteins can protect neuronal cells in culture and animals from WNV- induced death, we will correlation of neuronal survival with induction of calbindin D28k in the hamster model. We will also test if increased levels of calcium binding proteins from expression vectors will increase the survival of WNV-infected cells and rodents. We will establish a neuro-protection cell culture assay for evaluation of selective metabolites for induction of calcium buffer proteins, and for monitoring the survival of WNV-infected neuronal cells. Candidates will also be screened using a BBB transendothelial permeability assay. These assays will be necessary to select the most efficacious metabolite. The lead test agent will be evaluated in hamsters and in knockout transgenic mice. We will obtain preclinical data on more appropriate parenteral or oral treatment regimens in animal models. If there is evidence for animal efficacy of the lead metabolite, there is a possibility that dietary deficiency could correlate with severe neurological disease. Therefore, studies with deficient, normal and super levels of the metabolite in the diet of hamsters will be conducted. Also, serum samples from a cohort of 100 WNV patients will be assayed for the surrogate metabolite. In light of similarities of JEV with WNV, and that JEV is listed in the RFA instructions, the therapeutic calcium binding target will also be investigated fo JEV. Because of expertise in neurovirology, antiviral research, vitamin D metabolism, and calcium signaling, we are uniquely positioned to discover a therapy for viral encephalitis. This project could provide new avenues of basic research for WNV and JEV neuro-pathogenesis, and enhance clinical practice for the management of WNV, JEV, and viral encephalitis in general.