Molecular effects of metformin, PKR and TBI on C9orf72 ALS/FTD

二甲双胍、PKR 和 TBI 对 C9orf72 ALS/FTD 的分子效应

• 批准号: 10586260
• 负责人: Laura P.W Ranum
• 金额: $ 215.26万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586260
• 关键词: ALS patients; Adult; Affect; Antibodies; Autopsy; Bacterial Artificial Chromosomes; Behavior; Binding; Biochemical; Biological Assay; Brain; C9ORF72; Calorimetry; Cells; Clinical; Craniocerebral Trauma; Cyclic AMP-Dependent Protein Kinases; Data; Diabetes Mellitus; Diagnostic; Disease; Disease Outcome; Disease model; FDA approved; Family; Genes; Genetic; Genetic Transcription; Histopathology; In Vitro; Injury; Label; Manufactured football; Metformin; Military Personnel; Modeling; Molecular; Motor Neurons; Mus; Mutation; Organoids; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Play; Protein Kinase; Protein Region; Proteins; RNA; Recording of previous events; Research; Risk; Role; Series; Site; Stress; Stroke; TBI Patients; Testing; Transgenic Mice; Transgenic Organisms; Translations; Traumatic Brain Injury; Vulnerable Populations; Work; biological adaptation to stress; comparative; drug efficacy; effective therapy; experimental study; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genetic approach; genetic disorder diagnosis; improved; innovation; insight; prevent; protein expression; protein kinase P; protein kinase R; sporadic amyotrophic lateral sclerosis; therapeutic development; therapeutically effective; transcriptome sequencing

Project Summary The G4C2 repeat expansion mutation in the C9orf72 gene is the most common, known cause of familial and sporadic amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD)1,2. There are no effective treatments for C9-ALS/FTD or >50 other expansion disorders3,4. Our work identified two mechanisms now thought to play major roles in C9-ALS/FTD and other expansion diseases: 1) bidirectional transcription of expansion mutations5; and 2) repeat associated non-AUG (RAN) translation6. More than 90% of ALS presents as sporadic disease with no family history or genetic diagnosis. Surprisingly, our data show RAN proteins accumulate in ~44% of C9orf72-negative [C9(-)] sALS autopsy cases. The accumulation of toxic RAN proteins in C9(-) sALS and a growing number of other diseases highlights the need to develop drugs that block their production7-9. Activation of the integrated stress response (ISR) increases RAN translation10,11 and expansion RNAs activate the ISR kinase protein kinase R (PKR)12,13. We showed targeting RAN proteins in transgenic C9orf72 BAC mice with antibodies14 or PKR inhibition13 mitigates disease. We also discovered that metformin, a well-tolerated diabetes drug inhibits PKR, decreased RAN protein levels, improved behavior, histopathology, and motor neuron survival in C9-ALS/FTD mice13 but the mechanisms for these effects are not yet understood. These data combined with increased rates of ALS in patients with traumatic brain injury (TBI) suggest the possibility that stress and injury trigger ALS in vulnerable populations through increased RAN translation. This proposal aims to understand the mechanisms of PKR and metformin on RAN translation and to test TBI as a trigger of RAN translation and ALS that can be mitigated by PKR inhibition and metformin. In this proposal we will test the following hypothesis: (1) that metformin improves disease by reducing RAN proteins in C9orf72 ALS/FTD in a PKR dependent manner. Understanding the mechanism of action of metformin will provide important insights to advance its clinical use for C9-ALS/FTD and to improve drug efficacy; (2) that AAV-PKR-K296R is a highly effective therapeutic strategy to reduce RAN proteins in C9-ALS/FTD through p- eIF2α-dependent and independent pathways. Understanding how PKR regulates RAN translation and if AAV- PKR-K296R improves disease in adult C9-BAC mice will facilitate the development therapeutic strategies for C9orf72 ALS/FTD; and (3) that injury will increase RAN translation and exacerbate disease in C9orf72 mice and brain organoids from C9+ and genetically unknown, sporadic RAN+ sALS (gsr+ALS) patients and that reducing RAN protein levels will be protective. Taken together these innovative series of experiments could fundamentally change the diagnostics and treatment options for patients with C9-ALS, genetically unknown RAN positive sALS as well as the larger family of RAN protein associated expansions diseases. 1

项目摘要 C9orf72基因的G4C2重复扩增突变是家族性和家族性高血压最常见和已知的原因 散发性肌萎缩侧索硬化症和额颞叶痴呆(C9-ALS/FTD)1，2。 治疗C9-ALS/FTD或其他50种扩张性疾病3，4。我们的工作现在发现了两种机制 被认为在C9-ALS/FTD和其他扩张性疾病中起主要作用：1)双向转录 扩展突变5；以及2)重复相关的非8月(RAN)翻译6。90%以上的ALS患者 为散发性疾病，无家族病史或基因诊断。令人惊讶的是，我们的数据显示Ran蛋白质 在C9orf72阴性[C9(-)]尸检病例中积聚了约44%。有毒RAN蛋白的蓄积 在C9(-)SALS和越来越多的其他疾病中，强调了开发阻断其基因表达的药物的必要性 制作7胜9负。整合应激反应(ISR)的激活增加了RAN翻译10，11和扩展 RNA激活ISR蛋白激酶R(PKR)12，13。我们发现在转基因中靶向RAN蛋白 带有抗体14或PKR抑制13的C9ORF72 BAC小鼠可减轻疾病。我们还发现，二甲双胍是一种 耐受性良好的糖尿病药物抑制PKR，降低RAN蛋白水平，改善行为，组织病理学， 以及运动神经元在C9-ALS/FTD小鼠中的存活，但这些影响的机制尚不清楚。 这些数据结合创伤性脑损伤(TBI)患者肌萎缩侧索硬化症(ALS)发生率的增加，表明 应激和损伤通过增加RAN翻译在脆弱人群中触发ALS的可能性。这 该提案旨在了解PKR和二甲双胍对RAN翻译的作用机制，并测试TBI作为一种 RAN翻译和ALS的触发可通过PKR抑制和二甲双胍缓解。 在这项提案中，我们将检验以下假设：(1)二甲双胍通过减少射程来改善疾病 C9orf72 ALS/FTD中的蛋白以PKR依赖的方式表达。了解二甲双胍的作用机制 将为推进其在C9-ALS/FTD的临床应用和提高药物疗效提供重要的见解； AAV-PKR-K296R是一种高效的治疗策略，通过p-PKR-K296R可以减少C9-ALS/FTD中RAN蛋白的表达 EIF2依赖和独立的α通路。了解PKR如何规范RAN翻译，以及如果AAV- PKR-K296R改善成年C9-BAC小鼠疾病将促进制定治疗策略 C9ORF72 ALS/FTD；以及(3)这种损伤将增加C9orf72小鼠的RAN翻译并加剧疾病。 C9+和遗传未知的散发性RAN+ALS(GSR+ALS)患者的脑有机质及其减少 RAN蛋白水平将起到保护作用。综上所述，这些创新的系列实验可以从根本上 改变C9-ALS、遗传未知RAN阳性SAL患者的诊断和治疗选择 以及更大的RAN蛋白相关扩张性疾病家族。 1