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.

抽象的 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 影响大脑和脊髓各个区域运动神经元的疾病，对身体产生毁灭性影响 患者的健康和寿命。虽然研究已经确定了许多基因中的 ALS 和 FTD 突变（即 C9orf72、MAPT、SOD 和 GRN），大约 90% 的 ALS 和 60% 的 FTD 患者表现为散发性 遗传病因未知的病例（sALS 和 sFTD）。复杂的疾病机制加上大量的 遗传和表型异质的患者群体的研究和研究受到严重限制 这些疾病的治疗成功。内含子 C9orf72 G4C2 重复扩展突变的发现 由于 ALS 和 FTD 最常见的遗传原因将这些疾病与更大的微卫星家族联系起来 扩张障碍。这些疾病包括脊髓延髓肌萎缩症 (SBMA)，这是一种 CAG•CTG 疾病 与 ALS 和 FTD 一样，它也会影响运动神经元。据报道，越来越多的扩张障碍 通过重复相关的非 AUG (RAN) 翻译以多个阅读框表达蛋白质。我们最近的 未发表的研究结果表明，新型多聚丝氨酸和多聚亮氨酸 RAN 蛋白在十种中的至少六种中积累 CAG•CTG 多聚谷氨酸疾病。这些观察结果提出了新型多聚丝氨酸和多聚亮氨酸 RAN 的可能性 脊髓延髓肌萎缩症 (SBMA) 中积累的蛋白质和其他未识别的 RAN 蛋白质 可能会导致 sALS 和 sFTD。我们的中心假设是重复扩展突变表达 新型 RAN 蛋白在很大程度上导致 sALS、sFTD 和脊髓延髓肌萎缩症 (SBMA) 降低 RAN 蛋白水平的治疗方法将改善临床前模型中的疾病。致地址 根据这一假设，我们开发了一种创新的病理学到遗传学策略，可以快速、直接地 从患者 DNA 中鉴定产生扩展突变的新型 RAN 蛋白。我们很高兴报告 在初始筛查中，约 30% 的 sALS 尸检病例具有未知的遗传病因（即 C9 和 SCA36 GR 或 PR RAN 蛋白聚集体呈阳性，表明存在新的扩增 突变。在本提案中，我们将测试新型 RAN 蛋白有助于 sALS 的假设， sFTD 和 SBMA（目标 1）使用免疫测定以及患者血液和尸检组织样本。在目标 2 中，我们 将利用创新的 dCas9READ 方法来识别 RAN(+) sALS 中新的重复扩增突变， sFTD 病例并研究假定的致病扩展突变的毒性作用。最后我们来测试一下 使用 AAV-PKR(K296R) 或二甲双胍减少 RAN 翻译将改善疾病的假设 患者衍生的 sALS、sFTD 和 SBMA 诱导模型和小鼠模型中的表型（目标 3）。采取 这些研究将为 sALS、sFTD 和 SBMA 的分子机制提供重要见解 并促进开发统一的治疗方法来对抗这些毁灭性疾病。