Transfer RF1 AG054991 Beyond Haploinsuffiency- Gain of Function in Prograulin Mutations

转移 RF1 AG054991 超越单倍体不足 - Prograulin 突变的功能获得

• 批准号: 10399043
• 负责人: William Tzu-lung Hu
• 金额: $ 268.85万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10399043
• 关键词: Transfer; RF1; AG054991; Beyond; Haploinsuffiency

ABSTRACT Mutations in progranulin (GRN) represent one of the most common causes of familial frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). Progranulin is involved in inflammatory cascades, but the exact pathogenic link between GRN mutations and FTLD-TDP is unknown. Most mutations result in premature termination codons (PTC) in one GRN allele and 50% reduction on peripheral progranulin protein levels. However, this progranulin haploinsufficiency alone may not account for brain pathology as 1) progranulin deficiency precedes neurological symptom onset by decades, and 2) genetically reducing progranulin levels to 50% in mice does not produce significant pathologic or behavioral changes. During our investigation for inflammatory alterations in cerebrospinal fluid (CSF) from symptomatic GRN mutation carriers, we found different mutation groups to each lead to a cytokine profile. This led us to hypothesize that truncated mutant granulin peptides promote FTLD-TDP pathogenesis. Using sensitive RNA-Seq analysis, we confirmed that mutant GRN transcripts are detectable in brains at levels up to 20% of wildtype transcripts. We then used molecular modeling to design polyclonal antibodies targeting truncated R493X mutant progranulin peptide, and identified mutant progranulin dimers in brains and cultured fibroblasts of subjects carrying the same mutation. In the current application, we propose to extend our innovative findings in three aims by: 1) defining inflammatory phenotypes for GRN mutations predicted to result in short, intermediate, and long truncated mutant progranulin peptides; 2) confirming and characterizing mutant R493X peptides as dimers in carriers of this and other GRN mutations; and 3) directly testing whether mutant progranulin peptides derived from patient samples or plasmids can enhance the formation of TDP-43 oligomers. We will leverage the neurological, biochemical, neuropathological, and genetic expertise of the investigative team from Emory University and University of Pennsylvania, and resources from these two institutions as well as University of Brescia and the multi-centered Longitudinal Evaluation of Familial Frontotemporal Dementia Subjects (LEFFTDS). As an exploratory aim, we will also develop novel antibodies targeting two mutations predicted to result in intermediate-length progranulin peptides significantly shorter than R493X to generalize our findings. Upon completion of these aims, we will have performed clinical, pathological, and mechanistic analysis of the relationship between GRN mutations and FTLD-TDP to inform future therapeutic development and personalized diagnostics.

抽象的 前植物（GRN）中的突变代表家族性额颞叶的最常见原因之一 TDP-43夹杂物（FTLD-TDP）的变性。尿素蛋白参与炎症性级联反应，但 GRN突变与FTLD-TDP之间的确切致病联系尚不清楚。大多数突变导致过早 一个GRN等位基因中的终止密码子（PTC），外周素蛋白蛋白水平降低50％。 但是，仅这种易生蛋白单倍氨酸弥倍不足可能不会说明脑病理学，为1） 缺乏先于几十年的神经系统症状发作，而2）将遗传学水平降低至 50％的小鼠不会产生重大的病理或行为改变。在我们调查期间 有症状的GRN突变载体中脑脊液（CSF）的炎症改变，我们发现 每个突变组的不同突变导致细胞因子谱。这导致我们假设截断的突变体 颗粒蛋白肽促进FTLD-TDP发病机理。使用敏感的RNA-seq分析，我们证实了 突变的GRN转录本在大脑中可检测到高达20％的野生型转录本的水平。然后我们使用了 针对靶向截短的R493X突变体肽肽的分子建模，用于设计多克隆抗体， 在大脑和携带相同受试者的培养的成纤维细胞中鉴定出突变体二聚体二聚体 突变。在当前应用中，我们建议将我们的创新发现扩展到三个目标：1）定义 用于GRN突变的炎症表型预计会导致短，中间和长时间截断 突变体肽肽； 2）确认并将突变体R493X肽作为二聚体的载体 这个和其他GRN突变； 3）直接测试突变体易生蛋白肽是否衍生自患者 样品或质粒可以增强TDP-43低聚物的形成。我们将利用神经学， 埃默里大学（Emory University）和 宾夕法尼亚大学以及这两个机构的资源以及布雷西亚大学和 家族性额颞痴呆受试者（LEFFTDS）的多中心纵向评估。作为 探索目的，我们还将开发针对两个预测的突变的新型抗体 中间长度的前长肽肽明显短于R493X，以概括我们的发现。之上 这些目标的完成，我们将对 GRN突变与FTLD-TDP之间的关系，以告知未来的治疗发展和 个性化诊断。