Investigation of nuclear transport dysfunction in an ocular model of FTLD

FTLD 眼部模型中核转运功能障碍的研究

• 批准号: 8568516
• 负责人: Michael Emmerson Ward
• 金额: $ 22.51万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8568516
• 关键词: Address; Advisory Committees; Basic Science; Binding; Biochemical; Biological Assay; Blindness; Brain; Cell Aging; Cell Death; Cell Nucleus; Cells; Cellular biology; Cessation of life; Clinical; Complementary DNA; Cytoplasm; DNA; DNA-Binding Proteins; Data; Dementia; Development; Disease; Dominant-Negative Mutation; Environment; Exhibits; Eye; Fluorescence Resonance Energy Transfer; Frontotemporal Dementia; Functional disorder; Genes; Genetic Transcription; Goals; Grant; Guanosine Triphosphate; Imaging Techniques; Impaired cognition; Impairment; In Vitro; Individual; Institutes; Investigation; Knock-out; Knockout Mice; Lasers; Lead; Location; Mediating; Mentors; Mentorship; Messenger RNA; Microscopy; Modeling; Molecular; Molecular Biology Techniques; Monomeric GTP-Binding Proteins; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurology; Neuronal Dysfunction; Neurons; Nuclear; Nuclear Import; Ophthalmologist; Ophthalmoscopy; Organ; Pathway interactions; Patients; Phenotype; Progranulin; RNA Processing; RNA-Binding Proteins; Recombinants; Records; Reporting; Research; Research Personnel; Retina; Retinal; Retinal Ganglion Cells; Reverse Transcriptase Polymerase Chain Reaction; Running; Scanning; Scientist; Secondary to; Symptoms; Testing; Training; Transcription Process; Visual; Work; aged; alpha Karyopherins; base; career; cell age; cellular imaging; functional status; granulin; improved; in vivo; mouse model; mutant; neuronal survival; neuropathology; novel; novel diagnostics; novel therapeutics; nuclear transport factor 2; nucleocytoplasmic transport; progranulin protein; protein TDP-43; protein expression; public health relevance; research study; retinal nerve fiber layer; retinal neuron; skills; small hairpin RNA; therapeutic target; trafficking; visual information

Project Summary: Patients with dementing illnesses frequently complain of visual dysfunction, but given their cognitive dysfunction these symptoms may be overlooked. Though the cause of visual loss in dementing illnesses is poorly understood, it is likely that degeneration of the brain and eye share common molecular pathways. We discovered that patients with frontotemporal lobar dementia (FTLD), the most common cause of dementia in patients <60 years old, develop thinning of retinal nerve fiber layer (RNFL), indicating a loss of retinal ganglion cells (RGCs). We observed a similar phenotype in a mouse model of familial FTLD caused by loss of expression of the protein progranulin (PGRN). Using this new retinal model of FTLD, we are now able to probe previously inaccessible questions regarding FTLD pathophysiology. TDP-43 is a DNA/RNA binding protein that is normally concentrated in the nucleus. In many FTLD patients, nuclear depletion of TDP43 occurs and this loss contributes to neuron death. We found that RGCs from PGRN-knockout (KO) mice also exhibited nuclear depletion of TDP43. These cells also had impaired nuclear import of TDP43, and further study revealed that PGRN-KO RGCs with nuclear depletion of TDP43 also had loss of expression of the small GTPase Ran, a master regulator of nuclear transport. This proposal explores how loss of PGRN results in impaired Ran function, and how dysfunctional Ran- mediated nuclear transport leads to TDP43 mislocalization and RGC death. In Aim 1, I will use microscopy and molecular biology techniques to determine how PGRN loss disrupts Ran-mediated nuclear trafficking in RGCs. In Aim 2, I will use classic in vitro nuclear transport assays, expression approaches in primary RGCs, and in vivo rescue experiments to determine how abnormal Ran-mediated nuclear trafficking leads to TDP43 mislocalization and RGD death. A better understanding of these phenomena is a necessary first step in my long-term goal of developing novel therapies for patients with vision loss or dementia. I have a strong background in neurology and basic research and am thus well poised to carry out this project. The proposed training will take place jointly at the Gladstone Institutes and UCSF, both of which have long track records of training independent clinical-scientists. My primary mentor, Dr. Li Gan, is an expert at modeling neurodegenerative conditions in mice, and my co-mentor, Dr. Ari Green, is adept in retinal imaging techniques. I will augment their mentorship with an interdisciplinary advisory committee made up of a senior ophthalmologist (Dr. Stephen McLeod), experts in RGC biology and death (Drs. Erik Ullian and David Sretavan), an expert in the biochemical and molecular mechanisms of FTLD (Dr. Bob Farese), and a FTLD neuropathology expert (Dr. Bill Seeley). The skills gained through conducting the proposed studies in a mentored environment, combined with relevant coursework, professional development opportunities, and judicious subspecialty clinical training, will prepare me to launch my career as an independent investigator.

痴呆症患者经常抱怨视觉功能障碍，但考虑到他们的 认知功能障碍这些症状可能会被忽视。虽然痴呆症患者视力丧失的原因 虽然对这种疾病的了解很少，但很可能大脑和眼睛的退化有着共同的分子 途径。我们发现，额颞叶痴呆（FTLD）患者，最常见的原因， <60岁的痴呆患者，视网膜神经纤维层（RNFL）变薄，这表明 视网膜神经节细胞（RGCs）。我们观察到一个类似的表型在小鼠模型的家族性FTLD引起的 蛋白质颗粒蛋白前体（PGRN）的表达丧失。使用这种新的FTLD视网膜模型，我们现在能够 探索以前无法触及的关于FTLD病理生理学的问题。 TDP-43是一种DNA/RNA结合蛋白，通常集中在细胞核中。在许多FTLD患者中， 发生TDP 43的核耗竭，这种损失导致神经元死亡。我们发现， PGRN敲除（KO）小鼠也表现出TDP 43的核耗竭。这些细胞也有受损的核 进一步的研究显示，具有TDP 43核耗竭的PGRN-KO RGC也具有 核转运的主要调节因子小GTdR an的表达丧失。 该提案探讨了PGRN的丢失如何导致Ran功能受损，以及Ran功能障碍如何影响Ran的功能。 介导的核转运导致TDP 43错误定位和RGC死亡。在目标1中，我将使用显微镜， 分子生物学技术来确定PGRN损失如何破坏Ran介导的RGC中的核运输。 在目标2中，我将使用经典的体外核转运试验、原代RGC中的表达方法和体外细胞培养中的表达方法。 体内拯救实验，以确定异常Ran介导的核运输如何导致TDP 43 错误定位和RGD死亡。更好地理解这些现象是我的第一步。 长期目标是为视力丧失或痴呆患者开发新的治疗方法。我有一种强烈 我有神经病学和基础研究的背景，因此我准备好了执行这个项目。拟议 培训将在格莱斯顿研究所和加州大学旧金山分校联合进行，这两个机构都有长期的培训记录， 培养独立的临床科学家。我的主要导师李干博士是一位建模专家 我的同事阿里绿色博士擅长视网膜成像技术。 我将增加他们的指导与一个跨学科的咨询委员会组成的资深 眼科医生（Stephen McLeod博士），研究皇家地理学会生物学和死亡的专家（Erik Ullian和大卫博士 Sretavan），FTLD的生化和分子机制专家（Bob Farese博士），以及FTLD 神经病理学专家（比尔塞利博士）。通过开展拟议的研究获得的技能， 辅导环境，结合相关课程，专业发展机会，以及 明智的亚专业临床培训，将准备我开始我的职业生涯作为一个独立的调查员。