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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

基本信息

批准号：
8728869
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
项目类别：
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/8728869
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

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：痴呆症患者经常抱怨视力障碍，但考虑到他们的认知功能障碍，这些症状可能被忽视。虽然痴呆症中视力丧失的原因尚不清楚，但很可能大脑和眼睛的退化有共同的分子途径。我们发现，额颞叶痴呆（FTLD）是60岁以下患者中最常见的痴呆原因，其视网膜神经纤维层（RNFL）变薄，表明视网膜神经节细胞（RGCs）丢失。我们在家族性FTLD小鼠模型中观察到由蛋白前颗粒蛋白（PGRN）表达缺失引起的类似表型。利用这种新的视网膜FTLD模型，我们现在能够探索以前无法触及的关于FTLD病理生理的问题。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TDP43的核耗竭，这种损失导致神经元死亡。我们发现pgrn敲除（KO）小鼠的RGCs也表现出TDP43的核耗损。这些细胞也有TDP43的核输入受损，进一步的研究表明，TDP43核缺失的PGRN-KO RGCs也有小GTPase Ran的表达缺失，GTPase Ran是核运输的主要调节因子。本研究旨在探讨PGRN的缺失如何导致Ran功能受损，以及Ran介导的核转运功能障碍如何导致TDP43错定位和RGC死亡。在目标1中，我将使用显微镜和分子生物学技术来确定PGRN丢失如何破坏rgc中ran介导的核运输。在Aim 2中，我将使用经典的体外核转运试验、原发性RGCs的表达方法和体内救援实验来确定异常ran介导的核转运如何导致TDP43错定位和RGD死亡。更好地理解这些现象是我为视力丧失或痴呆症患者开发新疗法的长期目标的必要的第一步。我在神经学和基础研究方面有很强的背景，因此我很适合开展这个项目。拟议的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，这两所大学都有长期培训独立临床科学家的记录。我的主要导师李干博士是老鼠神经退行性疾病建模方面的专家，而我的共同导师阿里格林博士则擅长视网膜成像技术。我将与一个跨学科咨询委员会加强他们的指导，该委员会由一名高级眼科医生（Stephen McLeod博士）、研究资助局生物学和死亡方面的专家(dr。Erik Ullian和David Sretavan)， FTLD的生化和分子机制专家（Bob Farese博士）和FTLD神经病理学专家（Bill Seeley博士）。通过在有指导的环境中进行拟议的研究所获得的技能，结合相关课程，专业发展机会，以及明智的亚专业临床培训，将为我作为一名独立调查员的职业生涯做好准备。