Apoptotic nucleosomal DNA and its relevance in SLE

描述(由申请人提供)： 这份R03拨款提案旨在为我们的假设驱动项目提供“原则证明”，因此是NIH R01申请的坚实基础。 系统性红斑狼疮(SLE)是一种独特的致病自身抗体谱，主要针对普遍存在的核靶点。最常见的自身抗原是核小体，染色质的基本结构，以及它们的组成部分：双链DNA(DsDNA)和组蛋白。要了解系统性红斑狼疮的病因，了解核小体DNA的来源及其如何激发自身免疫反应是很重要的。在某些小鼠模型中，由于对凋亡细胞或其碎片的清除障碍可导致类SLE疾病，由于SLE自身抗原集中在凋亡的气泡和小体上，并且由于核小体DNA的产生是凋亡过程中最明显的特征之一，因此，凋亡细胞被认为是自身抗原的重要储存库。 我们打算使用一种基因工程小鼠，在这种小鼠中，DNA片段的能力被破坏，因此缺乏源于凋亡的核小体DNA，作为一种工具，在体内研究是否凋亡细胞是SLE核小体的主要来源。我们提出了三年的时间线，在三年内将实现两个目标。在AIM I中，我们将在没有caspase激活的DNA酶(CAD)的情况下，在体内研究染色质的重新分布、在体内和泡中的聚集以及在细胞凋亡过程中的释放。在AIM II中，我们建议建立几种小鼠狼疮模型，由于它们缺乏CAD，因此不能在凋亡过程中对DNA进行碎片化，因此缺乏源于凋亡的核小体DNA，以直接证明凋亡的核小体是SLE的驱动自身抗原，以及它们是发展和/或维持狼疮自身免疫反应所必需的。 AIM I和II的这组实验将为R01提案的应用提供初步的重要步骤，其长期目标是通过基因操作染色质碎裂来操纵狼疮疾病，并最终使用新技术在体内沉默CAD或其他基因，从而抑制凋亡核小体的产生，作为这种威胁生命的疾病的潜在治疗方法。

DESCRIPTION (provided by applicant): This R03 grant proposal is designed to provide the "proof of principle" of our hypothesis-driven project and is therefore the solid ground for an NIH R01 application. Systemic lupus erythematosus (SLE) is characterized by a unique spectrum of pathogenic autoantibodies, mainly directed at ubiquitous nuclear targets. The most frequent autoantigens are nucleosomes, the fundamental structure of chromatin, and their components: double stranded DNA (dsDNA) and histones. To understand the etiology of SLE, it is important to know the source of nucleosomal DNA and how it incites the autoimmune response. Because impaired clearance of apoptotic cells or their debris can lead to SLE-like disease in certain mouse models, because SLE autoantigens concentrate on apoptotic blebs and bodies, and because generation of nucleosomal DNA is one of the most distinct characteristics of the apoptotic process, apoptotic cells have been proposed as a critical reservoir of autoantigens. We intend to use a genetically engineered mouse in which the ability to fragment DNA is disrupted, and therefore nucleosomal DNA of apoptotic origin is absent, as a tool to investigate in vivo if apoptotic cells are the main source of nucleosomes in SLE. We propose a time line of three years in which 2 AIMs will be attained. In AIM I, we will investigate chromatin redistribution, concentration into bodies and blebs, and release during apoptosis in vivo, in the absence of caspase activated DNase (CAD), the most important known DNase involved in the generation of apoptotic nucleosomal DNA. In AIM II we propose to generate several murine lupus models, which "lack" nucleosomal DNA of apoptotic origin because they are CAD deficient, and therefore cannot fragment DNA during apoptosis, to directly demonstrate that apoptotic nucleosomes are the driving autoantigen in SLE, and that they are necessary to develop and/or sustain the autoimmune response in lupus. The sets of experiments in AIM I and II will provide the initial important steps toward an R01 proposal application, which long term goals are to genetically manipulate chromatin fragmentation in order to manipulate lupus disease, and ultimately to use novel technologies to silence in vivo CAD, or other genes and so inhibit the generation of apoptotic nucleosomes as a potential treatment of this life threatening disease.