Enhanced APOE2 Expression into Brain for Therapeutic Strategy for Alzheimer's Disease

摘要： 神经炎症是导致神经退行性疾病进展的主要危险因素之一。 中枢神经系统海马区新神经元的诞生贯穿一生， 神经发生的数量与海马区的学习记忆功能密切相关。 神经炎症单独抑制神经发生，并用抗炎剂阻断炎性 恢复神经再生。抗炎(AI)药物减少各种炎症细胞因子的表达 并增加突触数量。姜黄素、白藜芦醇、栎素和大麻二酚等天然药物 Origin具有抗炎活性和神经再生作用。APOE2、VGF和BDNF已被 被发现有助于在一生中维持细胞增殖、神经元功能和突触可塑性。此外， 已有研究表明，脑内APOE2、VGF和BDNF水平的升高与神经发生有关 和细胞增殖。因此，AI与APOE2或BDNF的结合将协同增强 神经发生和突触功能。所要求的补充奖的长期目标是设计一种 用于高效递送药物的非病毒基因递送载体，shAPOE4，编码APOE2，pAPOE2， 脑源性神经营养因子等基因可用于防治包括阿尔茨海默病在内的衰老相关的认知功能衰退。这个 所要求的补充设备奖将以下列方式加强药物发现的科学性： (1).人工智能药物在给药基质和生物体液中的测定以及体内药代动力学和 药物的高效液相生物分布：该系统将帮助我们量化人工智能药物的载药量 在纳米粒中，并在体内研究药物在不同器官中的药代动力学和生物分布。(2)。 实时定量聚合酶链式反应定量检测不同基质中的靶基因 聚合酶链式反应)、热循环仪和多模式微孔板阅读器：qRT-PCR提供了快速、高通量、可靠的 不同基质中靶基因的检测和定量。QRT-PCR仪器将使我们能够 量化作为时间函数的mRNA表达的变化。热循环器将允许转换为 将在qRT-PCR中进一步定量。Biotek™Synergy™HTX多模式微板 阅读器将在我们的实验室用于各种应用，从核酸定量，蛋白质 定量，酶动力学，生物标记物定量，ELISA，遗传分析，细胞增殖，细胞毒性， 以及药物的吸收和代谢。采购上述设备所需经费(190 981美元) 将有助于研究药物、mRNA、pAPOE2、shAPOE4、BDNF和其他核酸的传递 基于纳米颗粒的输送系统。我们预计所要求的设备将大大有助于 AD药物治疗的药物发现领域。因此，这些拟议的研究设备将 对开发以药物和核酸为基础的阿尔茨海默病治疗具有很高的影响。此外，所要求的设备 还可以向系内和大学的其他调查人员进行高质量的研究。

SUMMARY: Neuroinflammation is one of the major risk factor which leads to the progression of neurodegenerative disorders. The birth of new neurons within the hippocampal region of the central nervous system continues throughout life, and the amount of neurogenesis correlates closely with the hippocampal functions of learning and memory. Neuroinflammation alone inhibits neurogenesis and that inflammatory blockade with an anti-inflammatory agent restores neurogenesis. Anti-inflammatory (AI) drug reduces the expression of various inflammatory cytokines and increased synapse number. Drugs such as curcumin, resveratrol, quercetin and cannabidiol from natural origin have demonstrated both anti-inflammatory activity and neurogenesis. ApoE2, VGF and BDNF have been found useful in maintaining cell proliferation, neuronal function and synaptic plasticity throughout life. Moreover, it has been shown that an increased level of ApoE2, VGF, and BDNF in the brain is associated with neurogenesis and cell proliferation. Therefore combination of AI and ApoE2 or BDNF would synergistically enhance neurogenesis and synaptic functions. The long-term goal of the requested supplement award is to design a non-viral gene delivery carrier for efficient delivery of drugs, shAPOE4, mRNA encoding APOE2, pAPOE2, BDNF and other genes to brain for prevention and treatment of aging-related cognitive decile including AD. The requested supplement equipment award would strengthen the science of drug discovery in the following ways: (1).Determination of AI drugs in delivery matrix and biological fluids as well as in vivo pharmacokinetics and biodistributions of drugs by HPLC: The HPLC system would help us to quantify the amount of AI drugs entrapped in nanoparticles, and study in vivo the pharmacokinetics and biodistribution of drugs in various organs. (2). Quantification of target genes in different matrices by Quantitative Real Time Polymerase chain reaction (qRT- PCR), thermocycler, and multi-mode microplate reader: The qRT-PCR provides fast and high-throughput reliable detection and quantification of target genes in different matrices. The qRT-PCR instrument will enable us to quantify the changes in mRNA expression as a function of time. The thermocycler will allow the conversion of RNA to cDNA which will further be quantified in the qRT-PCR. BioTek™ Synergy™ HTX Multi-Mode Microplate Reader will be used for various applications in our lab ranging from nucleic acid quantification, protein quantification, enzyme kinetics, biomarker quantification, ELISAs, genetic analysis, cell proliferation, cytotoxicity, and drug absorption and metabolism. The requested fund ($190,981) to procure the above equipment items would help investigate delivery of drugs, mRNA, pAPOE2, shAPOE4, BDNF and other nucleic acids through nanoparticle-based delivery systems. We anticipate that the requested equipment will significantly contribute to the field of drug discovery for AD pharmacological therapy. Thus, these proposed research equipment would have high impact in developing drug and nucleic acid based therapy for AD. In addition, the requested equipment would also be available to conduct quality research to other investigators in the department and university.