Novel repeat associated non-AUG (RAN) proteins in sALS, sFTD and SBMA: shared pathological features and unifying therapeutic opportunities

sALS、sFTD 和 SBMA 中新型重复相关非 AUG (RAN) 蛋白：共同的病理特征和统一的治疗机会

• 批准号: 10420041
• 负责人: Lien Nguyen
• 金额: $ 193.01万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10420041
• 关键词: Address; Affect; Age of Onset; Amyotrophic Lateral Sclerosis; Antibodies; Antisense RNA; Autopsy; Bacterial Artificial Chromosomes; Blood; Brain; Brain region; C-terminal; C9ORF72; CASP8 gene; CRISPR/Cas technology; Case Study; Cell Culture Techniques; Cells; Climacteric; Clinical Data; Complex; Coupled; DNA; Data; Development; Diabetes Mellitus; Disease; Disease Management; Disease model; Dominant-Negative Mutation; FDA approved; Family; Family history of; Frontotemporal Dementia; Genes; Genetic; Genetic Predisposition to Disease; Genomic DNA; Health; Immunoassay; Immunohistochemistry; Immunologic Tests; Knowledge; Link; Longevity; Metformin; Methods; Microsatellite Repeats; Modeling; Molecular; Motor Neuron Disease; Motor Neurons; Mutation; Myotonic dystrophy type 2; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Organoids; Pathologic; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Polymers; Pre-Clinical Model; Proteins; RNA; Reading Frames; Reporting; Research; Sampling; Site; Spinal Cord; Spinobulbar Muscular Atrophy; Stress; Testing; Therapeutic; Tissue Sample; Tissues; Toxic effect; Transgenic Organisms; Translations; base; behavioral phenotyping; diagnostic tool; disease phenotype; effective therapy; experimental study; fighting; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genetic approach; improved; individual patient; innovation; insight; molecular phenotype; mouse model; neurodegenerative phenotype; neuroinflammation; novel; novel marker; novel strategies; novel therapeutics; patient population; protein aggregation; protein kinase R; response; spinal and bulbar muscular atrophy; sporadic amyotrophic lateral sclerosis; success; therapy development; tool; treatment strategy

ABSTRACT Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are complex neurodegenerative diseases that affect motor neurons in various regions of brain and spinal cord with devastating impacts on a patient’s health and lifespan. While research has identified ALS and FTD mutations in a number of genes (i.e. C9orf72, MAPT, SOD, and GRN), approximately 90% of ALS and 60% of FTD patients present as sporadic cases (sALS & sFTD) with unknown genetic etiology. Complex disease mechanisms coupled with a large genetically and phenotypically heterogeneous patient population have severely limited research and therapeutic success for these diseases. The discovery of the intronic C9orf72 G4C2 repeat expansion mutation as the most common genetic cause of ALS and FTD links these diseases to the larger family of microsatellite expansion disorders. Amongst these diseases is spinal-bulbar muscular atrophy (SBMA), a CAG•CTG disease that, like ALS and FTD, also affects motor neurons. A growing number of expansion disorders are reported to express proteins in multiple reading frames by repeat associated non-AUG (RAN) translation. Our recent unpublished findings show that novel polySer and polyLeu RAN proteins accumulate in at least six of the ten CAG•CTG polyGln diseases. These observations raise the possibility that novel polySer and polyLeu RAN proteins accumulate in the spinal-bulbar muscular atrophy (SBMA) and that other unidentified RAN proteins may contribute to sALS and sFTD. Our central hypothesis is that repeat expansion mutations that express novel RAN proteins substantially contribute to sALS, sFTD and spinobulbar muscular atrophy (SBMA) and that therapeutic approaches that reduce RAN protein levels will improve disease in preclinical models. To address this hypothesis, we have developed an innovative pathology-to-genetics strategy that enables rapid and direct identification of novel RAN protein producing expansion mutations from patient DNA. We are excited to report that in an initial screen, ~30% of sALS autopsy cases of unknown genetic etiology (i.e. C9 and SCA36 negative) were positive for GR or PR RAN protein aggregates suggesting the presence of novel expansion mutations. In this proposal, we will test the hypothesis that novel types of RAN proteins contribute to sALS, sFTD and SBMA (Aim 1) using immunoassays and patient blood and autopsy tissue samples. In Aim 2, we will utilize an innovative dCas9READ method to identify novel repeat expansion mutations in RAN(+) sALS and sFTD cases and study the toxic effects of putative disease-causing expansion mutations. Lastly, we will test the hypothesis that decreasing RAN translation using AAV-PKR(K296R) or metformin will improve disease phenotypes in patient derived induced models and mouse models of sALS, sFTD and SBMA (Aim 3). Taken together, these studies will provide critical insights into the molecular mechanisms of sALS, sFTD and SBMA and facilitate the development of unifying therapeutic approaches to fight these devastating diseases.

摘要 肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTD)是复杂的神经退行性疾病 影响大脑和脊髓不同区域运动神经元的疾病，对 患者的健康和寿命。虽然研究已经确定了一些基因(即ALS和FTD)的突变。 C9ORF72、MAPT、SOD和GRN)，大约90%的ALS和60%的FTD患者呈散发状态 遗传病因不明的SALS和sFTD 1例。复杂的疾病机制加上大量的 遗传和表型不同的患者群体严重限制了研究和 这些疾病的治疗取得了成功。内含子C9orf72 G4C2重复扩增突变的发现 作为ALS和FTD最常见的遗传原因，这些疾病与更大的微卫星家族有关 扩张性疾病。这些疾病包括脊髓性肌萎缩症(SBMA)，一种CAG·CTG疾病 这与肌萎缩侧索硬化症和FTD一样，也会影响运动神经元。据报道，越来越多的扩张性疾病 通过重复相关的非Aug(RAN)翻译在多个阅读框中表达蛋白质。我们最近 未发表的研究结果表明，新的PolySer和PolyLeu Ran蛋白至少在10个蛋白质中的6个中积累 CAG·CTG PolyGln病。这些观察结果增加了新的PolySer和PolyLeu范围内 蛋白质在脊髓性肌萎缩症(SBMA)中积聚，其他未知的RAN蛋白 可能导致SALS和sFTD。我们的中心假设是，表达的重复扩增突变 新的RAN蛋白在SALS、sFTD和脊髓延髓肌萎缩症(SBMA)中起重要作用，并且 降低RAN蛋白水平的治疗方法将改善临床前模型中的疾病。致信地址 这一假设，我们开发了一种创新的病理学到遗传学的策略，使快速和直接 从患者DNA中鉴定产生扩张性突变的新的RAN蛋白。我们很高兴地向大家报告 在初步筛查中，约30%的遗传病因不明的SALS尸检病例(即C9和SCA36 阴性)均为GR或PR RAN蛋白聚集体阳性，提示存在新的扩张 突变。在这个提议中，我们将检验新类型的RAN蛋白对SALS有贡献的假设， SFTD和SBMA(AIM 1)使用免疫分析以及患者血液和尸检组织样本。在目标2中，我们 将利用一种创新的dCas9READ方法在RAN(+)SAL和 SFTD病例，并研究可能致病的扩展突变的毒性效应。最后，我们将测试 使用AAV-PKR(K296R)或二甲双胍减少RAN翻译将改善疾病的假说 SALS、sFTD和SBMA患者诱导模型和小鼠模型的表型(目标3)。已被占用 总之，这些研究将对sals、sftd和sbma的分子机制提供重要的见解。 并促进制定统一的治疗方法，与这些毁灭性的疾病作斗争。