The role of a novel atypical monoamine transporter in Alzheimer's disease

描述(由申请人提供)：在动物模型和患者中，淀粉样多肽(A？)的升高和积聚是阿尔茨海默病(AD)的显著病理特征。淀粉样前体蛋白(APP)的产生是由两种蛋白水解酶--β-位裂解酶(BACE1)和β-分泌酶催化的。最近的研究发现，APP的BACE1裂解位点附近有一种突变，使那些携带该突变的人对AD和与年龄相关的认知能力下降具有抵抗力。这表明BACE1介导的APP切割对AD风险和疾病进展具有重要意义，并验证了介导BACE1活性进行干预的前景。为了寻找BACE1的细胞调控因子，我们使用了一种化学遗传学的方法来寻找能够靶向影响BACE1切割APP的新途径的化学探针。一种检测特定BACE1介导的APP裂解产物--分泌型APP？(Sapp？)的方法被用于在细胞平台中筛选化学探针。确定了一种结构类似于中枢神经系统(CNS)中活性药物的结构类似的命中化合物。为了提高疗效和探索构效关系，随后合成了能够降低Sapp？和A？的化学类似物(CNS系列)。然而，CNS系列的特定细胞靶点仍然未知。基于文献描述的母体化合物的药物类别、已报道的脑内可能靶点的区域表达以及广泛的药理学测试，我们确定了一个候选靶点--质膜单胺转运体(PmAt)。PMAT是一种非典型的单胺转运蛋白，属于一类高度可药物的单胺转运蛋白。在啮齿类动物中，pmAt在大脑中的表达，这些区域被证明是AD的影响区域，包括内嗅皮层和海马体。我们的初步研究表明，从药物上抑制pmAt并从基因上减少pmAt在神经元中的表达会导致Sapp？和A？的水平降低。有趣的是，用pmAt抑制剂处理神经元会导致AMPK(AMPK)哺乳动物的雷帕霉素靶点(MTOR)信号通路中蛋白质的翻译后改变。我们建议在小鼠模型和人类中验证pmAt对AD的影响的假设，并确定对Sapp？？和A？生物发生的影响的细胞和分子机制。这些研究的成功完成将证实pmAt是AD的一个新的细胞靶点，有可能被用于未来AD治疗的药物开发。我近期的职业目标是研究pmAt在小鼠AD发病机制中的作用，以及它对人类AD病理的潜在贡献。这些目标的实现将有助于我的长期研究目标，即成为一名有效和多产的独立调查者，为AD研究领域做出重大贡献。以我在生物化学和药理学方面的背景，我希望将我的专业知识扩展到AD的翻译研究中，包括靶点验证和人类病理学。有了这个培训奖，我将能够发展转运蛋白生物学方面的专业知识，以及人类 AD的病理学。哥伦比亚大学病理学和细胞生物系的共同导师(Kim博士和Shelanski博士)和环境是实现这些目标的唯一合适人选。此外，我还将把合作者、来自华盛顿大学的pmat专家王博士纳入我在哥伦比亚大学之外的科研网络。哥伦比亚大学的环境充满了合作者，包括但肯定不限于贾维奇博士和冯萨特尔博士，这促进了我的科学和职业发展。为了促进独立，我计划向NIH提交后续R01资金的申请。在准备过程中，我将参加哥伦比亚大学欧文临床与翻译研究所的第一堂R01课程。我还将通过参加哥伦比亚大学提供的正式课程来发展生物统计学和转运蛋白生物学方面的技能。哥伦比亚大学还提供广泛的研讨会，吸引专家科学家，促进讨论和合作。此外，准备未来教师研讨会系列和学术事务办公室教师发展系列提供关于职业发展技能的研讨会，如助学金撰写、手稿准备和演示。我还有一流的资源，可以用来进行负责任的研究行为培训。最后，我建议通过参加戈登研究研讨会和会议、冷泉港实验室会议以及阿尔茨海默氏症协会国际会议，包括阿尔茨海默氏症国际会议和阿尔茨海默氏症和帕金森氏病国际会议，获得转运体生物学和细胞信号级联方面的培训。这些机会中的每一个都将使我能够发展特定的知识以及我的职业关系网，这是在科学领域取得成功的关键组成部分。发展成为一名成功的独立调查员需要勤奋的研究规划和执行、特殊的培训和个人和机构的关键支持，所有这些都在本申请中得到了例证。完成所建议的科学和培训部分，将使我成为AD领域的一名高技能和公认的贡献者。在我作为该领域的成功贡献者不断发展的过程中，它还将促进随后的、尽管LSS正式的、职业生涯长的独立发展。

DESCRIPTION (provided by applicant): Elevation and accumulation of amyloid ß-peptide (Aß) are hallmark pathological characteristics of Alzheimer's disease (AD) in animal models and patients. The production of Aß is mediated by cleavage of amyloid precursor protein (APP) by two proteolytic enzymes, ß-site cleaving enzyme (BACE1) and ?- secretase. Recent work has identified a mutation in close proximity to the BACE1 cleavage site of APP rendering those who bear the mutation resistant to AD and age-associated cognitive decline. This indicates BACE1 mediated cleavage of APP is seminal for AD risk and disease progression, and validates the promise of mediating BACE1 activity for intervention. To find cellular regulators of BACE1, we employed a chemical genetics approach to identify chemical probes that that can target novel pathways which effect BACE1 cleavage of APP. An assay for detecting the specific BACE1 mediated APP cleavage product, secreted APPß (sAPPß), was used to screen for chemical probes in a cell-based platform. A hit compound was identified which was structurally similar to a well characterized drug class active in the central nervous system (CNS). To improve the efficacy and explore the structure activity relationship, chemical analogs were subsequently synthesized (CNS-series) which were able to reduce sAPPß and Aß. However, the specific cellular target of the CNS-series remained unknown. Based on literature describing the drug class of the parent compound, the reported regional expression of putative targets in brain and extensive pharmacological testing, we identified a candidate target, the plasma membrane monoamine transporter (PMAT). PMAT is an atypical monoamine transporter belonging to a highly druggable class of monoaomine transporters. In rodents, PMAT is expressed in the brain in regions shown to be affected in AD including entorhinal cortex and hippocampus. Our preliminary studies indicated that pharmacologically inhibiting PMAT and genetically reducing PMAT expression in neurons resulted in decreased sAPPß and Aß levels. Interestingly, treatment of neurons with PMAT inhibitors led to post-translational alterations of proteins in AMP-activated protein kinase (AMPK) mammalian target of rapamycin (mTOR) signaling pathways. We propose to test the hypotheses that PMAT contributes to AD in a mouse model and in human and identify the cellular and molecular mechanisms contributing to the effect on sAPPß and Aß biogenesis. Successful completion of these studies will validate PMAT as a novel cellular target in AD which has potential to be pharmacologically harnessed for future development of AD therapeutics. My immediate career goals are to study the role of PMAT in pathogenesis of AD in a mouse model and its potential contribution to AD pathology in human. Achievement of these goals will facilitate my long-term research goal which is to become an effective and productive independent investigator to contribute significantly to the field of AD research. With my background in biochemistry and pharmacology, I hope to expand my expertise into translational studies of AD including target validation and human pathology. With this training award I will be able to develop expertise in transporter biology as well as the human pathology of AD. The co-mentors (Drs. Kim and Shelanski) and environment in the Department of Pathology and Cell Biology at Columbia University are uniquely suited for achievement of these goals. Additionally, I will expand my scientific network outside of Columbia University by including collaborator, Dr. Wang, a PMAT expert from the University of Washington. The environment at Columbia University is rich with collaborators including but certainly not limited to Drs. Javitch and Vonsattel, facilitating my scientific and career development. To facilitate independence, I plan on submitting an application for subsequent R01 funding from NIH. In preparation, I will take the Irving Institute for Clinical and Translational Research at Columbia University, Reach for the First R01 course. I also will develop skills in biostatistics and transporter biology by partaking in formal coursework offered by Columbia University. Columbia University also offers extensive seminars bringing expert scientists and facilitating discussions and collaborations. Additionally, the Preparing Future Faculty Seminar Series and Office of Academic Affairs Faculty Development Series offers seminars on career development skills such as grant writing, manuscript preparation and presentations. I also have superlative resources for training in Responsible Conduct of Research. Finally, I propose to gain training in both transporter biology and cell signaling cascades through attendance at Gordon Research Seminars and Conferences, Cold Spring Harbor Laboratory Meetings as well as international meetings in including Alzheimer's Association International Conference and International Conference on Alzheimer's disease and Parkinson's disease. Each of these opportunities will allow me to develop specific knowledge as well as my career network which are critical components for success in the field of science. Development into a successful independent investigator requires diligent research planning and execution, exceptional training and critical support, personally and institutionally, all of which are exemplified in this application. Completin of both the scientific and training portions proposed will enable my emergence as a highly skilled and recognized contributor to the field of AD. lt will also facilitate subsequent, though lss formal, career-long independent development during my continual evolution as a successful contributor to the field.