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

摘要 原颗粒蛋白(GRN)突变是家族性额颞叶的最常见原因之一 TDP-43夹杂物退化(FTLD-TDP)。原颗粒参与炎症性级联反应，但 GRN突变与FTLD-TDP之间确切的致病联系尚不清楚。大多数突变都会导致早产 一个GRN等位基因的终止密码子(PTC)和外周血前颗粒蛋白水平降低了50%。 然而，这种原颗粒单倍性不足本身可能不能解释原颗粒那样的大脑病理 缺乏比神经系统症状出现早几十年，以及2)基因上将原颗粒蛋白水平降低到 50%的小鼠不会产生明显的病理或行为变化。在我们调查 我们发现症状性GRN突变携带者脑脊液(CSF)中的炎性改变 不同的突变群会导致细胞因子图谱。这让我们假设被截短的突变体 颗粒多肽促进FTLD-TDP的发病。使用敏感的RNA-Seq分析，我们证实 突变型GRN转录本在大脑中可检测到高达20%的野生型转录本。然后我们使用 分子建模以设计针对截短的R493X突变体前颗粒蛋白多肽的多克隆抗体，以及 在脑和培养的携带前颗粒二聚体的受试者的成纤维细胞中发现突变的原颗粒二聚体 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突变的炎性表型预测会导致短、中、长截断 突变型前颗粒蛋白多肽；2)突变型R493X多肽作为载体二聚体的确认和鉴定 这个GRN突变和其他GRN突变；以及3)直接检测突变的原颗粒蛋白多肽是否来自患者 样品或质粒可促进TDP-43寡聚体的形成。我们将利用神经学的， 来自埃默里大学的研究小组的生化、神经病理学和遗传学专业知识 宾夕法尼亚大学，以及来自这两个机构以及布雷西亚大学和 家族性额颞叶痴呆患者的多中心纵向评估作为一种 探索性的目的，我们还将开发针对两个突变的新抗体，预计将导致 中间长度的原颗粒多肽明显短于R493X，以推广我们的发现。vt.在.的基础上 完成这些目标后，我们将进行临床、病理和机制分析 GRN突变与FTLD-TDP的关系为未来的治疗开发和 个性化诊断。

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.