Investigation of nuclear transport dysfunction in an ocular model of FTLD

描述（由申请人提供）：患有痴呆症的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。虽然痴呆症中视力丧失的原因还不清楚，但大脑和眼睛的退化可能有共同的分子途径。我们发现，额颞叶痴呆（FTLD）是<60岁患者痴呆的最常见原因，其患者出现视网膜神经纤维层（RNFL）变薄，表明视网膜神经节细胞（RGC）丢失。我们观察到一个类似的表型在小鼠模型的家族性FTLD引起的蛋白质前颗粒蛋白（PGRN）的表达损失。使用这种新的FTLD视网膜模型，我们现在能够探索以前无法解决的关于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的核耗竭。这些细胞还具有受损的TDP43的核输入，并且进一步的研究揭示了具有TDP43的核消耗的PGRN-KO RGC也具有小GTCRan Ran（核转运的主调节物）的表达缺失。 该提议探索了PGRN的损失如何导致Ran功能受损，以及功能失调的Ran介导的核转运如何导致TDP43错误定位和RGC死亡。在目标1中，我将使用显微镜和分子生物学技术，以确定如何PGRN的损失破坏Ran介导的核运输RGC。在目标2中，我将使用经典的体外核转运试验，在原代RGCs的表达方法，并在体内救援实验，以确定如何异常RAN介导的核运输导致TDP43的错误定位和RGD死亡。更好地理解这些现象是我为视力丧失或痴呆患者开发新疗法的长期目标的必要的第一步。我在神经病学和基础研究方面有很强的背景，因此很好地准备开展这个项目。拟议中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，这两个研究所都有培训独立临床科学家的长期记录。我的主要导师Li Gan博士是小鼠神经退行性疾病建模专家，我的共同导师Ari绿色博士擅长视网膜成像技术。我将通过一个跨学科咨询委员会来加强对他们的指导，该委员会由一名资深眼科医生（斯蒂芬·麦克劳德博士）、RGC生物学和死亡方面的专家（埃里克·尤里安博士和大卫·斯雷塔万博士）、FTLD生化和分子机制方面的专家（鲍勃·法雷博士）和FTLD神经病理学专家（比尔·塞利博士）组成。通过在辅导环境中进行拟议的研究所获得的技能，结合相关的课程，专业发展机会和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研究者。

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.