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

项目摘要/摘要 研究：人脑包含100多种不同类别和亚类的神经元和神经胶质细胞，这使得 体内最异质的器官。最近的研究表明，在衰老过程中，脑细胞 积累具有反映不同诱变因素的模式的体细胞突变。然而，到目前为止，这些 研究一直无法系统地描述特定个体的衰老相关体细胞突变率(SMR) 由于缺乏从人脑组织中分离特定细胞类型的方法而导致的脑细胞类型 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从尸检中分离出潜在的任何细胞类型来填补这一知识空白 人类大脑，在避免固定以保持高保真DNA基因组的完整性的同时- SMR的测序(DNA-seq)和分析。了解SMR和突变签名有何不同 在大脑中的细胞类型之间是重要的，因为它是评估 通常涉及特定细胞类型的与年龄相关的脑部疾病的体细胞突变和基因组完整性。这个 该提案的目的是：1)开发一种新技术，从冰冻的细胞中分离出任何类型的未固定的细胞核 尸检人脑，一种可能也适用于任何组织的方法；2)确定 SMR跨越年龄跨度，针对与年龄相关的脑病相关的四种特定的人类脑细胞类型。 这项提议的成功完成将使未来对体细胞突变和其他分子的研究成为可能 在不同年龄的背景下，通过固定潜在的任何细胞类型和大脑区域而排除的侧写- 相关的脑部疾病。培训计划：在这个奖项下，我将1)扩大我的科学知识 突变和基因组完整性的机制，以及在人脑老化的背景下；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)从事职业 为成功过渡到未来的独立学术研究事业而开展的发展活动。 环境：拟议的研究和培训计划将在吉拉德·埃弗罗尼博士的实验室进行， 纽约大学格罗斯曼医学院人类遗传学和基因组学中心(CHGG)医学博士 (NYUSOM)，由中心主任Aravinda Chakravarti博士共同指导。NYUSOM是一个 与受人尊敬的、致力于学员成功的老牌研究小组合作，创造良好的环境。埃弗罗尼医生是 是单细胞基因组学和计算基因组学的先驱和专家，提供严格和支持的 研究环境和职业指导。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，谁将提供进一步的指导科学，计算和职业生涯 目标。这两个实验室，合作的研究社区，以及我们与神经科学和 系统遗传学研究所，提供一个独特的环境来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.