Modulating the Nucleopathy caused by FUS Mutants of ALS

调节 ALS FUS 突变体引起的核病

基本信息

批准号：
9270636
负责人：
LAWRENCE J HAYWARD
金额：
$ 20.94万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9270636
关键词：
Acute Promyelocytic Leukemia Age Aging Amyotrophic Lateral Sclerosis Biological Assay Blood - brain barrier anatomy Cell Aging Cell Line Cell Nucleus Cell model Cells Cellular Stress Chemicals Chronic Collaborations DNA Damage Defect Development Dose Employee Strikes Engineering Exposure to FDA approved Familial Amyotrophic Lateral Sclerosis Fibroblasts Financial compensation Functional disorder Genes Genetic Genetic Transcription Homeostasis Human Image Impairment Laboratories Libraries Link Mediating Motor Motor Neurons Mus Muscle Mutation Nerve Nerve Degeneration Neurodegenerative Disorders Neuroglia Neuronal Injury Neurons Nuclear Nuclear Matrix Nuclear Protein Oxidative Stress Pathogenesis Pathology Pathway interactions Pharmaceutical Preparations Phenotype Preparation Proteasome Inhibition Proteins Quality Control RNA Processing RNA-Binding Protein FUS Reporting Resolution Role Stress Structure Testing Tissues Transcriptional Regulation Transgenic Mice Transgenic Organisms Validation Variant age related base biological adaptation to stress cellular imaging effective therapy experimental study high throughput screening injured link protein medical schools middle age monolayer multicatalytic endopeptidase complex mutant neuron loss novel novel therapeutic intervention novel therapeutics nucleic acid binding protein premature protein misfolding proteostasis repaired response screening small molecule success

项目摘要

Abstract Dominant mutations in the gene encoding the nucleic acid binding protein FUS cause ~5% of familial amyotrophic lateral sclerosis (ALS). Our long-term objectives are to discern the mechanism(s) by which FUS mutants injure aging motor neurons and to develop novel therapeutic approaches to increase the defenses against these insults. Our laboratory has identified a novel and robust nuclear phenotype caused by ALS- linked FUS mutants: impaired stress-responsive processing of sub-nuclear assemblies known as promyelocytic leukemia (PML) nuclear bodies. PML nuclear bodies are induced by a variety of cellular stresses and regulate nuclear protein homeostasis, transcription, DNA-damage pathways, and cellular senescence, yet their potential role in ALS has not been explored. We observed that PML bodies were abnormally enlarged both in cell lines and in primary ALS human fibroblasts expressing mutant FUS. Furthermore, proteasome activities were decreased, and exposure to mild oxidative stress or proteasome inhibition in FUS mutant but not control cells stalled the turnover of expanded PML bodies. We hypothesize that the observed abnormality of PML nuclear bodies may report upon altered nuclear homeostasis resulting from FUS mutant expression. In Aim 1 of this project, we will develop an imaging-based phenotypic screening assay to identify small molecule compounds that modulate the observed PML nuclear body enlargement in cells expressing FUS mutants. We will test compound libraries that include FDA-approved drugs and diverse CNS-active agents predicted to cross the blood-brain barrier. We will prioritize initial hits using dose-response studies and will determine whether proteins known to be targeted to the proteasome following stress are more effectively eliminated upon treatment with hit compounds. We have established transgenic mice harboring ALS-linked FUS variants and have observed a phenotype of age-dependent loss of the connection between motor nerves and muscle in mice that express the R495X mutant. In Aim 2 of this project, we will validate hit compounds and analyze pathways related to PML nuclear body function in CNS cells and tissues from our mutant FUS transgenic mice. We will test whether a subset of the prioritized hit compounds from Aim 1 ameliorate defects of nuclear protein homeostasis or proteasome activity in transgenic primary cortical neurons, glial cells, or motor neurons. In further experiments, which do not depend upon the success of obtaining modulator compounds in Aim 1, we will define more precisely which pathways related to PML nuclear body function are most relevant to FUS- mediated ALS. We will purify proteasomes from the CNS of our FUS mice at pre-symptomatic and symptomatic ages and will quantify subunit expression, assembly, and activities in collaboration with Dr. Fred Goldberg's group. We will also use a high-resolution tissue monolayer preparation to identify nuclear insults related to altered PML body function in aging neurons from our FUS transgenic mice.

抽象的编码核酸结合蛋白 FUS 的基因的显性突变导致约 5% 的家族性遗传病肌萎缩侧索硬化症（ALS）。我们的长期目标是辨别 FUS 的机制突变体损伤老化的运动神经元并开发新的治疗方法来增强防御反对这些侮辱。我们的实验室已经鉴定出一种由 ALS 引起的新颖且强大的核表型- 连锁 FUS 突变体：称为早幼粒细胞的亚核组件的应激反应加工受损白血病（PML）核小体。 PML 核体由多种细胞应激诱导并调节核蛋白稳态、转录、DNA 损伤途径和细胞衰老，但它们的潜力在 ALS 中的作用尚未被探讨。我们观察到细胞系中的 PML 小体异常增大以及表达突变 FUS 的原发性 ALS 人成纤维细胞。此外，蛋白酶体活性 FUS 突变体细胞减少，并且暴露于轻度氧化应激或蛋白酶体抑制，但对照细胞则不然阻碍了扩大的 PML 机构的更替。我们假设观察到的 PML 核异常身体可能会报告因 FUS 突变体表达而导致的核稳态改变。在这个目标1中项目中，我们将开发一种基于成像的表型筛选方法来鉴定小分子化合物调节表达 FUS 突变体的细胞中观察到的 PML 核体增大。我们将测试化合物库，其中包括 FDA 批准的药物和多种中枢神经系统活性药物，预计将跨越血脑屏障。我们将使用剂量反应研究优先考虑初始打击，并确定是否已知在应激后靶向蛋白酶体的蛋白质在用命中化合物进行治疗。我们已经建立了携带 ALS 相关 FUS 变体的转基因小鼠，观察到小鼠运动神经和肌肉之间连接的年龄依赖性丧失的表型表达 R495X 突变体。在该项目的目标 2 中，我们将验证命中化合物并分析路径与我们突变型 FUS 转基因小鼠的 CNS 细胞和组织中的 PML 核体功能相关。我们将测试来自 Aim 1 的优先命中化合物的子集是否可以改善核蛋白缺陷转基因原代皮质神经元、神经胶质细胞或运动神经元中的稳态或蛋白酶体活性。在进一步的实验，不依赖于目标 1 中调节剂化合物的成功获得，我们将更准确地定义哪些与 PML 核体功能相关的途径与 FUS 最相关介导的 ALS。我们将在症状发生前和症状发生前从 FUS 小鼠的中枢神经系统中纯化蛋白酶体。症状年龄，并将与 Fred 博士合作量化亚基表达、组装和活动戈德堡小组。我们还将使用高分辨率组织单层制剂来识别核损伤与 FUS 转基因小鼠衰老神经元 PML 身体功能改变有关。