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 C9 orf 72基因中的G4 C2重复扩增突变是最常见的、已知的家族性和遗传性疾病的原因。 散发性肌萎缩侧索硬化和额颞叶痴呆（C9-ALS/FTD）1，2.没有有效 C9-ALS/FTD或>50种其他扩张性疾病的治疗3，4。我们的工作现在确定了两种机制 被认为在C9-ALS/FTD和其他扩张性疾病中起主要作用：1） 扩增突变5;和2）重复相关的非AUG（RAN）扩增6。超过90%的ALS患者 没有家族史或基因诊断的散发性疾病。令人惊讶的是，我们的数据显示RAN蛋白 在~44%的C9 orf 72阴性[C9（-）] sALS尸检病例中累积。有毒RAN蛋白的积累 在C9（-）sALS和越来越多的其他疾病中， 生产7 -9。整合应激反应（ISR）的激活增加了RAN抑制10，11和扩张 RNA激活ISR激酶蛋白激酶R（PKR）12，13。我们在转基因小鼠中发现了靶向RAN蛋白， 具有抗体14或PKR抑制13的C9 orf 72 BAC小鼠减轻疾病。我们还发现二甲双胍， 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， C9-ALS/FTD小鼠中的运动神经元存活率13，但这些作用的机制尚不清楚。 这些数据结合创伤性脑损伤（TBI）患者ALS发生率的增加表明， 压力和伤害可能通过增加RAN翻译在弱势群体中引发ALS。这 该提案旨在了解PKR和二甲双胍对RAN翻译的机制，并将TBI作为一种 触发RAN翻译和ALS，可通过PKR抑制和二甲双胍缓解。 在本提案中，我们将检验以下假设：（1）二甲双胍通过降低RAN改善疾病 C9 orf 72 ALS/FTD中的蛋白质。了解二甲双胍的作用机制 将为推进C9-ALS/FTD的临床应用和提高药物疗效提供重要见解;（2） AAV-PKR-K296 R是一种通过p-磷酸化减少C9-ALS/FTD中RAN蛋白的高效治疗策略。 eIF 2 α依赖性和非依赖性途径。了解PKR如何调节RAN翻译，如果AAV- PKR-K296 R改善成年C9-BAC小鼠的疾病将促进开发治疗策略， C9 orf 72 ALS/FTD;和（3）损伤将增加RAN翻译并加重C9 orf 72小鼠的疾病， 来自C9+和遗传学未知的散发性RAN+ sALS（gsr+ALS）患者的脑类器官， RAN蛋白水平将是保护性的。这些创新的系列实验可以从根本上 改变C9-ALS患者的诊断和治疗选择，遗传学未知的RAN阳性sALS 以及更大的RAN蛋白相关扩张疾病家族。 1