Investigation of nuclear transport dysfunction in an ocular model of FTLD

项目摘要：痴呆症患者经常抱怨视觉功能障碍，但鉴于他们的 认知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 人们对疾病知之甚少，很可能是大脑和眼睛的退化共享共同的分子 小路。我们发现额颞叶痴呆(FTLD)患者是 60岁的痴呆症患者发展为视网膜神经纤维层变薄(RNFL)，表明 视网膜神经节细胞(RGC)。我们在家族性FTLD小鼠模型中观察到了类似的表型，该模型由 原颗粒蛋白(PGRN)表达缺失。使用FTLD的这种新的视网膜模型，我们现在能够 探讨以前难以解决的FTLD病理生理学问题。 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， TDP43发生核耗竭，这种丢失导致神经元死亡。我们发现RGC来自 PGRN基因敲除(KO)小鼠也表现出TDP43的核耗竭。这些细胞也有受损的核。 TDP43的导入，进一步的研究发现，TDP43核耗竭的PGRN-KO RGC也有 核运输的主要调节因子小GTP酶RAN的表达缺失。 该方案探讨了PGRN丢失如何导致RAN功能受损，以及RAN功能障碍是如何导致RAN功能受损的。 介导的核转运导致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PGRN丢失如何扰乱RAN介导的RGC核贩运。 在目标2中，我将使用经典的体外核转运分析，原代视网膜节细胞的表达方法，以及 活体救援实验确定异常RAN介导的核转运如何导致TDP43 本地化错误和RGD死亡。更好地理解这些现象是我在 为视力丧失或痴呆症患者开发新疗法的长期目标。我有一个很强的 我有神经学和基础研究的背景，因此我准备好执行这个项目。建议数 培训将在格拉德斯通学院和加州大学旧金山分校联合进行，这两个学院都有长期的 培养独立的临床科学家。我的主要导师李干博士是一位模特专家 我的另一位导师Ari Green博士精通视网膜成像技术。 我将通过一个由一名学长组成的跨学科咨询委员会来加强他们的指导 眼科医生(斯蒂芬·麦克劳德博士)，RGC生物学和死亡专家(埃里克·乌利安和大卫博士 在FTLD的生化和分子机制方面的专家(Bob Farese博士)和FTLD 神经病理学专家(Bill Seeley博士)。通过将拟议的研究在 有指导的环境，结合相关课程、职业发展机会，以及 明智的专科临床培训，将为我开始作为一名独立调查员的职业生涯做好准备。

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.