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

摘要 颗粒蛋白前体（GRN）的突变是家族性额颞叶疾病最常见的病因之一。 TDP-43包涵体变性（FTLD-TDP）。颗粒蛋白前体参与炎症级联反应，但 GRN突变和FTLD-TDP之间确切的致病联系尚不清楚。大多数突变导致过早 终止密码子（PTC）在一个GRN等位基因和50%的外周颗粒蛋白前体蛋白水平下降。 然而，这种颗粒蛋白前体单倍不足可能不能解释脑病理学， 缺乏症先于神经症状发作数十年，和2）遗传性降低颗粒蛋白原水平， 50%的小鼠不会产生显著的病理或行为变化。在我们调查 我们发现，有症状的GRN突变携带者的脑脊液（CSF）中存在炎症改变， 不同的突变组各自导致细胞因子谱。这使我们假设截短的突变体 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， 突变GRN转录物在脑中的检测水平高达野生型转录物的20%。然后我们使用 设计靶向截短的R493 X突变颗粒蛋白前体肽的多克隆抗体的分子建模，和 在脑中鉴定突变颗粒蛋白前体二聚体和携带该二聚体的受试者的培养成纤维细胞 突变在本申请中，我们提出通过以下三个目的来扩展我们的创新发现：1）定义 GRN突变的炎症表型预测会导致短、中、长截短的 突变体颗粒蛋白前体肽; 2）确认和表征突变体R493 X肽在载体中的二聚体， 这种和其他GRN突变;和3）直接测试是否来源于患者的突变颗粒蛋白前体肽 样品或质粒可以增强TDP-43寡聚体的形成。我们会利用神经学， 来自埃默里大学的调查小组的生物化学、神经病理学和遗传学专业知识， 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 为了探索性的目的，我们还将开发针对两种突变的新型抗体， 中等长度的颗粒蛋白前体肽显著短于R493 X以概括我们的发现。后 完成这些目标，我们将进行临床，病理和机制分析， GRN突变和FTLD-TDP之间的关系，以告知未来的治疗开发， 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.