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 颗粒体蛋白前体 (GRN) 突变是家族性额颞叶最常见的原因之一 TDP-43 内含物变性 (FTLD-TDP)。颗粒体蛋白前体参与炎症级联反应，但 GRN 突变与 FTLD-TDP 之间的确切致病联系尚不清楚。大多数突变会导致早产 一个 GRN 等位基因中的终止密码子 (PTC) 和外周颗粒体蛋白前体蛋白水平降低 50%。 然而，这种颗粒体蛋白前体单倍体不足可能无法单独解释脑病理学，因为 1) 颗粒体蛋白前体 缺乏症会在神经系统症状出现前数十年出现，并且 2) 从基因角度将颗粒体蛋白前体水平降低至 50%在小鼠中不会产生显着的病理或行为变化。在我们调查期间 我们发现有症状的 GRN 突变携带者的脑脊液 (CSF) 发生炎症改变 不同的突变组各自导致细胞因子谱。这使我们推测截短的突变体 颗粒蛋白肽促进 FTLD-TDP 发病机制。使用灵敏的 RNA-Seq 分析，我们证实 大脑中可检测到突变型 GRN 转录物，其水平高达野生型转录物的 20%。然后我们使用 分子模型设计针对截短的 R493X 突变颗粒体蛋白前肽的多克隆抗体，以及 在携带相同基因的受试者的大脑和培养的成纤维细胞中鉴定出突变的颗粒体蛋白前体二聚体 突变。在当前的应用中，我们建议通过以下三个目标扩展我们的创新发现：1）定义 GRN 突变的炎症表型预计会导致短、中、长截短 突变颗粒体蛋白前体肽； 2) 确认和表征突变体 R493X 肽作为载体中的二聚体 这种突变和其他 GRN 突变； 3) 直接检测突变颗粒体蛋白前体肽是否源自患者 样品或质粒可以增强TDP-43寡聚体的形成。我们将利用神经学、 埃默里大学研究小组的生化、神经病理学和遗传专业知识 宾夕法尼亚大学，以及这两个机构以及布雷西亚大学和 家族性额颞叶痴呆受试者的多中心纵向评估（LEFFTDS）。作为一个 为了探索性目标，我们还将开发针对预计会导致的两种突变的新型抗体 中等长度的颗粒体蛋白前体肽明显短于 R493X，以概括我们的发现。之上 为了完成这些目标，我们将对这些疾病进行临床、病理和机制分析。 GRN 突变和 FTLD-TDP 之间的关系，为未来的治疗开发提供信息 个性化诊断。