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.

项目总结 这项研究的首要目标是使用互补的果蝇和可诱导的多能干细胞 (IPSC)衍生的神经元模型以了解Ubiquin 2(UBQLN2)基因突变是如何导致 肌萎缩侧索硬化症/额颞叶痴呆(ALS/FTD)。UBQLN2及其密切相关的UBQLN1 属于真核泛素(Ub)结合蛋白家族，其部分功能是作为蛋白质的伴侣 它们注定会被蛋白酶体降解。唯一的功能性孤儿中的错义突变 UBQLN2的Proline-Repeat区域(PRR)导致X连锁的ALS/FTD，一些患者表现出 纯粹的痴呆症发作。此外，泛素组织病理学，由UBQLN2和UBQLN2的致密聚集体组成 无论UBQLN1突变状态如何，在大多数ALS/FTD病例中都能观察到UBQLN1。致信地址 UBQLN2相关FTD的发病机制我们利用了上游激活序列(UAS)/GAL4 表达野生型(WT)和ALS突变型UBQLN2的果蝇等基因菌株的产生系统 在不同的组织和细胞类型中。我们发现UBQLN2ALS突变体诱导出剂量依赖的表型- 包括眼睛退化、运动缺陷和寿命缩短--这些都比表型更严重 由UBQLN2WT的等价表达引起。形成了UBQLN2ALS突变体，但没有形成UBQLN2WT ALS/FTD患者中发现的泛素内包涵体的神经元内聚集体特征。无偏基因 筛选确定了30多个降低或增强UBQLN2ALS突变毒性的遗传间隔。 基因定位研究表明，内溶酶体途径在UBQLN2ALS介导中起中心作用 神经退行性变，并指向轴突引导基因和神经元依赖受体 新型疾病修饰剂。在这份R01拨款提案中，我们将继续对UBQLN2介导的基因进行研究 果蝇的神经退行性变，以内溶酶体转运和轴突引导为主导途径 用于探索的努力。在果蝇中发现的修饰通路将反过来为IPSC来源的研究提供信息 运动神经元表达内源性UBQLN2ALS等位基因。目标1的具体目标是：(A) 调查UBQLN2ALS果蝇的Rab5基因和内溶酶体缺陷；(B)调查 UNC-5轴突引导通路到UBQLN2介导的神经退行性变；以及(C)映射和验证 果蝇中的UBQLN2ALS修饰基因。目标2具体目标是：(A)建立职能部门 UBQLN2ALS iMN的缺陷；(B)进行UBQLN2ALS iMN的蛋白质组学分析；以及(C)评估基因 从果蝇中筛选出AIM 1作为iMN中UBQLN2ALS毒性的遗传修饰物。合并后的 利用果蝇和哺乳动物的IMN模型进行遗传、细胞和生化研究将提供重要的 对ALS/FTD中UBQLN2相关神经退行性变的新见解。此外，在我们的 这项研究可能会与其他聚集倾向的ALS/FTD- 相关基因。