Measurement of cell-type specific somatic mutation rates in the aging human brain - Resubmission – 1

项目概要/摘要 研究：人类大脑包含超过100种不同的神经元和神经胶质类和子类，使其 身体中最不均匀的器官。最近的研究表明，在衰老过程中， 积累体细胞突变的模式，反映了不同的诱变因素。然而，迄今为止，这些 研究一直无法系统地分析特定的衰老相关的体细胞突变率（SMR）， 由于缺乏从人脑组织中分离特定细胞类型的方法， 固定过程，这排除了测量SMR所需的高保真测序。这项建议 旨在通过开发一种方法来填补这一知识空白，该方法可以从死后组织中分离出任何类型的细胞， 人类大脑，同时避免固定，以保持高保真DNA基因组的完整性- 测序（DNA-seq）和SMR的分析。了解SMR和突变签名的区别 在大脑中的细胞类型是重要的，因为它是评估的可能作用的先决条件， 体细胞突变和基因组完整性与年龄相关的脑部疾病，往往涉及特定的细胞类型。的 该提案的目标是：1）开发一种新的技术，从冷冻细胞中分离任何类型的未固定细胞核。 死后的人脑，一种也可能适用于任何组织的方法; 2）为了确定 与年龄相关脑部疾病相关的四种特定人脑细胞类型的跨年龄段的SMR。 这项提案的成功完成将使未来的体细胞突变和其他分子生物学研究成为可能。 在不同年龄的背景下，通过固定在潜在的任何细胞类型和大脑区域中而排除的分析- 相关的大脑疾病。培训计划：在这个奖项下，我将1）扩大我的科学知识， 一般的诱变和基因组完整性的机制，以及在衰老人脑的背景下; 2） 获得基因组学和DNA-seq分析方面的计算和生物信息学专业知识; 3）开展职业生涯 发展活动，以成功过渡到未来的独立学术研究生涯。 环境：拟议的研究和培训计划将在Gilad Evrony博士的实验室进行， 纽约大学格罗斯曼医学院人类遗传学和基因组学中心（CHGG）的医学博士、博士 （NYUSOM），与该中心主任Aravinda Chakravarti博士共同指导。NYUSOM是一个 环境与备受尊敬的和建立的研究小组致力于学员的成功。埃夫隆尼医生 单细胞基因组学和计算基因组学的先驱和专家，提供严格的和支持性的 研究环境沿着职业导师。查克拉瓦蒂博士是著名的人类遗传学专家 和基因组学，以及统计学，谁将提供科学，计算和职业生涯的进一步指导， 目标.这两个实验室，合作研究社区，以及我们与神经科学和 系统遗传学研究所，提供了一个独特的环境，整合神经科学和基因组学培训， 并扩大科学和职业发展活动，以帮助我向独立过渡。

Project summary/Abstract Research: The human brain contains over 100 diverse classes and subclasses of neurons and glia, making it the most heterogenous organ in the body. Recent studies have revealed that during aging, brain cells accumulate somatic mutations with patterns that reflect diverse mutagenic factors. However, to date, these studies have been unable to systematically profile aging-related somatic mutation rates (SMRs) of specific brain cell types due to the lack of methods to isolate specific cell types from human brain tissue without the process of fixation, which precludes high-fidelity sequencing necessary for measuring SMRs. This proposal aims to fill this knowledge gap by developing a method to isolate potentially any cell type from post-mortem human brain, while avoiding fixation in order to preserve the integrity of the genome for high-fidelity DNA- sequencing (DNA-seq) and analysis of SMRs. Understanding how SMRs and mutational signatures differ among cell types in the brain is important, because it is a prerequisite for assessing the possible role of somatic mutations and genome integrity in age-related brain diseases that often involve specific cell types. The proposal’s aims are: 1) To develop a novel technology to isolate unfixed nuclei of any cell type from frozen post-mortem human brains, a method which will also potentially be applicable to any tissue; 2) To determine SMRs across age spans for four specific human brain cell types relevant to age-related brain diseases. Successful completion of this proposal will enable future studies of somatic mutations and other molecular profiling that is precluded by fixation in potentially any cell type and brain region in the context of diverse age- related brain disorders. Training Plan: Under this award, I will 1) Expand my scientific knowledge of the mechanisms of mutagenesis and genome integrity generally, and in the context of the aging human brain; 2) Gain computational and bioinformatics expertise in genomics and DNA-seq analysis; 3) Conduct career development activities for successful transition towards a future independent academic research career. Environment: The proposed research and training plans will be conducted in the laboratory of Dr. Gilad Evrony, MD, PhD, at the Center for Human Genetics and Genomics (CHGG) at NYU Grossman School of Medicine (NYUSOM), with co-mentorship from the Center’s director, Dr. Aravinda Chakravarti, PhD. NYUSOM is an environment with well-respected and established research groups dedicated to trainees’ success. Dr. Evrony is a pioneer and expert in single-cell genomics and computational genomics, providing a rigorous and supportive research environment along with career mentorship. Dr. Chakravarti is a renowned expert in human genetics and genomics, as well as statistics, who will provide further guidance on scientific, computational, and career goals. Both laboratories, the collaborating research community, and our affiliations with the Neuroscience and Systems Genetics Institutes, provide a unique environment to integrate neuroscience and genomics training, and to expand scientific and career-advancement activities to aid my transition towards independence.