Genetic analysis of UBQLN2-associated neurodegeneration in frontotemporal dementia

额颞叶痴呆中UBQLN2相关神经变性的遗传分析

• 批准号: 10157746
• 负责人: Randal Scot Tibbetts
• 金额: $ 170万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10157746
• 关键词: Address; Aging; Alleles; Alzheimer' s disease related dementia; Amyotrophic Lateral Sclerosis; Apoptosis; Applications Grants; Attenuated; Autopsy; Axonal Transport; Binding; Binding Proteins; Biochemical; Cell Culture Techniques; Cell Line; Characteristics; Chromosome Mapping; Chromosomes; Clinical; Colon Carcinoma; Cytology; Defect; Dementia; Dementia With Amyotrophic Lateral Sclerosis; Dependence; Development; Disease; Disease Pathway; Dissection; Dose; Drosophila genus; Drosophila melanogaster; Enhancers; Exhibits; Eye; Family; Frontotemporal Dementia; Functional disorder; Future; Gene Mutation; Genes; Genetic; Genetic Screening; Genetic study; Goals; Heat Stress Disorders; Histopathology; Human; Lead; Link; Longevity; Mammalian Cell; Maps; Mediating; Missense Mutation; Modeling; Molecular Chaperones; Motor; Motor Neurons; Mutation; Natural Immunity; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Junction; Neurons; Neuropathogenesis; Orphan; Parkinson Disease; Pathogenesis; Pathologic; Pathway interactions; Patients; Phenotype; Photoreceptors; Pluripotent Stem Cells; Process; Production; Proline; Property; Proteins; Proteomics; Stress; System; Testing; Tissues; Toxic effect; UBQLN1 gene; Ubiquitin; Up-Regulation; axon guidance; cell type; drug development; fly; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genetic analysis; induced pluripotent stem cell; insight; interest; mutant; mutational status; neuron apoptosis; neuronal survival; novel; protein aggregation; protein degradation; protein folding; protein function; proteostasis; proteotoxicity; receptor; stem cells; trafficking; transcriptomics; ubiquilin

Project summary The overarching goal of this study is to use complementary Drosophila and inducible pluripotent stem cells (iPSC)-derived neuronal models to understand how mutations in the Ubiquilin 2 (UBQLN2) gene cause amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). UBQLN2 and closely related UBQLN1 belong to a family of eukaryotic ubiquitin (Ub)-binding proteins that function, in part, as chaperones for proteins that are destined for proteasomal degradation. Missense mutations within a unique, functionally orphan proline-repeat region (PRR) of UBQLN2 cause X-linked, forms of ALS/FTD, with some patients exhibiting a pure dementia onset. In addition, ubiquilin histopathology, comprised of dense aggregates of UBQLN2 and UBQLN1 are observed in most instances of ALS/FTD regardless of UBQLN2 mutation status. To address pathomechanisms of UBQLN2-associated FTD we exploited the upstream activating sequence (UAS)/GAL4 system to generate isogenic Drosophila strains expressing wild-type (WT) and ALS mutant forms of UBQLN2 in different tissues and cell types. We found that UBQLN2ALS mutants elicited dose-dependent phenotypes— including eye degeneration, motor defects, and lifespan shortening—that were more severe than phenotypes caused by equivalent expression of UBQLN2WT. UBQLN2ALSmutants, but not UBQLN2WT, formed intraneuronal aggregates characteristic of ubiquilin inclusions found in ALS/FTD patients. Unbiased genetic screens identified more than 30 genetic intervals that either reduced or enhanced UBQLN2ALS mutant toxicity. Gene mapping studies suggest that endolysosomal pathways are centrally involved in UBQLN2ALS-mediated neurodegeneration and point toward axon guidance genes and neuronal dependence receptors as potentially novel disease modifiers. In this R01 grant proposal we will continue genetic studies of UBQLN2-mediated neurodegeneration in Drosophila, focusing on endolysosomal trafficking and axon guidance as lead pathways for discovery efforts. Modifier pathways identified in Drosophila will, in turn, inform studies of iPSC-derived motor neurons (iMNs) expressing endogenous UBQLN2ALS alleles. The specific objectives of Aim 1 are to: (a) investigate the Rab5 gene and endolysosomal defects in UBQLN2ALS flies; (b) investigate the contributions of the Unc-5 axon guidance pathway to UBQLN2-mediated neurodegeneration; and (c) map and validate of UBQLN2ALS modifier genes in Drosophila. The specific objectives of Aim 2 are to: (a) establish functional defects in UBQLN2ALS iMNs; (b) carry out proteomic analysis of UBQLN2ALS iMNs; and (c) evaluate genes emerging from Drosophila screens in Aim 1 as genetic modifiers of UBQLN2ALS toxicity in iMNs. The combined genetic, cellular, and biochemical studies using Drosophila and mammalian iMN models will provide important new insights into UBQLN2-associated neurodegeneration in ALS/FTD. In addition, pathways identified in our study are likely to overlap with and inform toxicity pathways instigated by other aggregation-prone, ALS/FTD- associated genes.

项目摘要 这项研究的首要目标是利用互补的果蝇和诱导性多能干细胞 （iPSC）衍生的神经元模型，以了解Ubiquilin 2（UBQLN 2）基因突变如何导致 肌萎缩侧索硬化/额颞叶痴呆（ALS/FTD）。UBQLN 2和密切相关的UBQLN 1 属于真核泛素（Ub）结合蛋白家族，部分功能为蛋白质伴侣 这些蛋白酶体被蛋白酶体降解。在一个独特的，功能性孤儿中的错义突变 UBQLN 2的脯氨酸重复区（PRR）引起X连锁形式的ALS/FTD，一些患者表现出 纯粹的痴呆发作此外，泛素组织病理学，包括UBQLN 2的密集聚集体和 无论UBQLN 2突变状态如何，在大多数ALS/FTD病例中均观察到UBQLN 1。解决 UBQLN 2相关FTD的病理机制，我们利用上游激活序列（UAS）/GAL 4 产生表达野生型（WT）和ALS突变体形式UBQLN 2的同基因果蝇菌株的系统 在不同的组织和细胞类型中。我们发现UBQLN 2ALS突变体引起剂量依赖性表型- 包括眼睛退化、运动缺陷和寿命缩短--这些比表型更严重 由UBQLN 2 WT的等效表达引起。形成了UBQLN 2ALS突变体，但未形成UBQLN 2 WT ALS/FTD患者中发现的泛素包涵体特征性神经元内聚集体。无偏遗传 筛选确定了超过30个降低或增强UBQLN 2ALS突变毒性的遗传间隔。 基因定位研究表明，内溶酶体途径主要参与UBQLN 2ALS介导的 神经变性和指向轴突导向基因和神经元依赖受体作为潜在的 新型疾病调节剂。在这个R 01拨款提案中，我们将继续进行UBQLN 2介导的遗传研究。 果蝇的神经退行性变，重点是内溶酶体运输和轴突引导作为主要途径 用于探索工作。在果蝇中发现的修饰途径反过来将为iPSC衍生的研究提供信息。 运动神经元（iMN）表达内源性UBQLN 2ALS等位基因。目标1的具体目标是： 研究UBQLN 2ALS果蝇中Rab 5基因和内溶酶体缺陷;（B）研究 UBQLN 2介导神经变性的Unc-5轴突引导途径;和（c）绘制和验证 果蝇中的UBQLN 2ALS修饰基因。目标2的具体目标是： UBQLN 2 ALS iMN中的缺陷;（B）进行UBQLN 2 ALS iMN的蛋白质组学分析;以及（c）评估基因 作为iMN中UBQLN 2ALS毒性的遗传修饰剂出现在Aim 1中的果蝇筛选中。将合并的 使用果蝇和哺乳动物iMN模型进行遗传、细胞和生化研究，将提供重要的 ALS/FTD中UBQLN 2相关神经变性的新见解。此外，在我们的研究中发现的途径 研究可能与其他聚集倾向、ALS/FTD引发毒性途径重叠， 相关基因