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

描述（由申请人提供）：淀粉样蛋白β-肽（Aβ）的升高和积累是阿尔茨海默病（AD）动物模型和患者的标志性病理特征。Aβ的产生是通过β位点切割酶（BACE1）和γ分泌酶（γ-secretase）两种蛋白水解酶对淀粉样蛋白前体蛋白（APP）的裂解介导的。最近的研究发现，在APP的BACE1切割位点附近有一个突变，使携带该突变的人能够抵抗AD和与年龄相关的认知能力下降。这表明BACE1介导的APP裂解对AD风险和疾病进展具有重要意义，并验证了通过介导BACE1活性进行干预的前景。为了找到BACE1的细胞调节因子，我们采用化学遗传学方法来鉴定可以靶向影响BACE1切割APP的新途径的化学探针。通过检测BACE1介导的特定APP切割产物分泌的APPβ (sAPPβ)，我们在基于细胞的平台上筛选化学探针。鉴定出一种hit化合物，其结构类似于一种在中枢神经系统（CNS）中具有活性的已被充分表征的药物类别。为了提高药效，探索其构效关系，随后合成了能降低sAPPβ和Aβ的化学类似物（cns系列）。然而，cns系列的具体细胞靶点尚不清楚。根据文献描述母体化合物的药物类别，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，即质膜单胺转运蛋白（PMAT）。PMAT是一种非典型的单胺转运蛋白，属于高度可药物化的单胺转运蛋白。在啮齿类动物中，PMAT在阿尔茨海默病中受影响的大脑区域表达，包括内嗅皮质和海马。我们的初步研究表明，药物抑制PMAT和基因降低PMAT在神经元中的表达可导致sAPPβ和Aβ水平下降。有趣的是，用PMAT抑制剂处理神经元会导致amp活化的蛋白激酶（AMPK）哺乳动物雷帕霉素靶蛋白（mTOR）信号通路中蛋白的翻译后改变。我们建议在小鼠模型和人类模型中验证PMAT对AD的影响，并确定影响sAPβ和a β生物发生的细胞和分子机制。这些研究的成功完成将验证PMAT作为阿尔茨海默病的一个新的细胞靶点，有可能在药理学上用于阿尔茨海默病治疗的未来开发。我的近期职业目标是研究PMAT在小鼠AD发病机制中的作用及其对人类AD病理的潜在贡献。这些目标的实现将促进我的长期研究目标，即成为一名有效和富有成效的独立研究者，为AD研究领域做出重大贡献。凭借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的转化研究，包括靶标验证和人类病理学。有了这个培训奖，我将能够在转运体生物学和人类方面发展专业知识

DESCRIPTION (provided by applicant): Elevation and accumulation of amyloid β-peptide (Aβ) are hallmark pathological characteristics of Alzheimer's disease (AD) in animal models and patients. The production of Aβ is mediated by cleavage of amyloid precursor protein (APP) by two proteolytic enzymes, β-site cleaving enzyme (BACE1) and γ-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 APPβ (sAPPβ), 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 sAPPβ and Aβ. 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 sAPPβ and Aβ 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 sAPβ and Aβ 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.