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

摘要 肌萎缩侧索硬化（ALS）和额颞叶痴呆（FTD）是一种复杂的神经退行性疾病， 影响大脑和脊髓各区域运动神经元的疾病， 患者的健康和寿命。虽然研究已经确定了ALS和FTD在许多基因中的突变（即， C9 orf 72、MAPT、SOD和GRN），约90%的ALS患者和60%的FTD患者表现为散发性 例（sALS和sFTD），遗传病因不明。复杂的疾病机制加上大规模的 遗传和表型异质的患者群体严重限制了研究， 这些疾病的治疗效果。内含子C9 orf 72 G4 C2重复扩增突变的发现 作为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， 扩张障碍这些疾病中有脊髓延髓肌萎缩症（SBMA），一种CAG·CTG疾病， 就像ALS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蛋白质在10种蛋白质中的至少6种中积累， CAG·CTG polyGln疾病。这些观察结果提出了新的polySer和polyLeu RAN 蛋白质积累在脊髓延髓肌萎缩症（SBMA）和其他不明RAN蛋白 可能导致sALS和sFTD。我们的中心假设是，重复扩增突变，表达 新的RAN蛋白实质上导致sALS、sFTD和脊髓延髓肌萎缩（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。解决 基于这一假设，我们开发了一种创新的病理学-遗传学策略， 从患者DNA鉴定产生扩增突变的新RAN蛋白。我们很高兴地报告 在初始筛选中，约30%的sALS尸检病例的遗传病因不明（即C9和SCA 36 阴性）对GR或PR RAN蛋白聚集体呈阳性，表明存在新的扩增 突变。在这项提案中，我们将测试新类型的RAN蛋白有助于sALS的假设， 使用免疫测定法和患者血液和尸检组织样本测定sFTD和SBMA（目的1）。在目标2中， 将利用创新的dCas 9 READ方法来鉴定RAN（+）sALS中的新型重复扩增突变， sFTD病例，并研究推定致病扩展突变的毒性作用。最后，我们将测试 使用AAV-PKR（K296 R）或二甲双胍减少RAN翻译将改善疾病的假设 患者诱导模型和sALS、sFTD和SBMA小鼠模型中的表型（目的3）。采取 总之，这些研究将对sALS、sFTD和SBMA的分子机制提供重要的见解 并促进统一治疗方法的发展，以对抗这些毁灭性的疾病。

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 Our recent unpublished findings show that novel polySer and polyLeu RAN proteins accumulate in at least six of the ten CAG•CTG polyGln diseases.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.