SINGLE-CELL ANALYSIS OF SOMATIC MUTATION IN AGING AND NEUROEGENERATIVE DISEASE IN THE HUMAN BRAIN

项目摘要/摘要 候选人。我对了解基因和表观遗传机制有极大的兴趣 细胞状态，并在我的研究生涯中建立了这种兴趣。作为一名本科生研究员， 霍夫斯特拉大学乔安妮·威利博士的实验室，我研究了细菌芽胞形成和抗生素生产 使用遗传学和扫描电子显微镜，导致了同行评议的手稿的作者身份。作为一名 在麻省理工学院Rudolf Jaenisch博士的实验室攻读博士学位时，我在 在几个导致同行评审手稿作者的项目中，与表观遗传学有关的因素-- 介导性重编程和转分化。在我的主要论文工作中，我使用了基因组学，遗传学， 分子生物学和组织培养技术，以研究细胞命运在发育中的转变 哺乳动物神经系统，导致第一作者发表(Lodato等人，PLoS Genetics，2013，PMID 23437007)。在我博士后研究的第一部分，在波士顿克里斯托弗·沃尔什博士的实验室里 儿童医院和哈佛医学院，我在所有这些经验的基础上，特别是在 用基因组学理解神经生物学原理，研究正常人的体细胞突变 大脑使用尖端的单细胞全基因组测序(WGS)技术，导致了第一作者 手稿(Lodato等人，《科学》，2015年，PMID 26430121)。这篇论文是第一篇发表于 比较正常人类个体的全基因组频率和体细胞突变特征， 而我开发这项技术所获得的专业知识，让我完全准备好了现在可以比较 以及年龄组之间和人类年龄相关疾病中的体细胞突变的特点。 K99/R00独立之路奖是帮助我推动我的 科学和职业目标。根据这个奖项，我将执行一项研究计划，以揭示躯体的作用 衰老和与年龄相关的疾病中的突变2)获得科学技能并加深我对关键 老龄化和生物信息学领域的概念3)在以下情况下开展职业发展活动 我的导师、合作导师和其他教职顾问致力于我成功地从 从博士后研究员到独立调查员。 环境拟议的研究和培训计划将在克里斯托弗·A·A的实验室进行。 沃尔什，医学博士，波士顿儿童医院(BCH)和哈佛医学院(HMS)。这些 各机构组成了一个强大的、成熟的研究社区，并致力于 本申请中提出的目的和目标。沃尔什博士在培训博士后方面有很好的记录 研究员，他以前的许多实习生现在学术学院担任终身教职或终身教职 机构。这一成功源于实验室严谨和支持的环境，包括频繁的实验室 与沃尔什博士的会面和一对一的互动。从科学上讲，实验室提供了几乎所有的 拟议研究所需的试剂和设备，并作为更大的BCH和HMS的一部分 社区可在步行距离内使用任何附加设备。BCH/HMS社区也是 这是该提案在职业发展方面的一项资产，提供了与潜在合作者的轻松联系 经常举办研讨会，主持内部和外部演讲者。作为这个社区的一部分，我已经获得了一个 共同导师(Peter Park博士，HMS)和另外两名教职顾问(HMS Bruce Yanker博士和Emanuela Gussoni，BCH/HMS)，都致力于我的成功。最后，BCH/HMS环境提供了无数正式的 职业发展研讨会和研讨会，我将参加这些研讨会和研讨会，帮助我过渡到独立。 研究。在所有器官系统中，高龄是人类疾病的主要风险因素，然而 衰老现象影响的组织范围如此之广，目前尚不清楚。所有的组织都依赖于 基因组才能正常运作，而一个有吸引力且长期存在的假设是， DNA损伤的积累可能是衰老的原因。这一概念的证据在大脑中仍然难以捉摸， 由于标准的DNA测序实验不适合检测体细胞突变，而体细胞突变可能只标志着 在一个由数百万个细胞组成的样本中，有几个细胞，甚至一个细胞。我是使用单细胞的先驱， 全基因组测序技术比较体细胞性疾病的发生率、特征和后果 人类大脑中的突变，并将使用这项技术作为该奖项的一部分，以实现三个目标：1) 确定衰老是否与人脑中突变的积累有关，并确定 衰老过程中的体细胞突变模式，2)与老年性疾病相关的单细胞测序 DNA损伤反应，以及3)检测阿尔茨海默病的体细胞突变率。因此，这一点 提案代表了一项全面的工作，将阐明体细胞突变在体内的作用 衰老，加速衰老，以及人脑中与年龄相关的疾病。

Project Summary/Abstract Candidate. I have a profound interest in understanding the genetic and epigenetic mechanisms that control cell state, and have built upon that interest during my research career. As an undergraduate researcher in the laboratory of Dr. Joanne Wiley at Hofstra University, I studied bacterial sporulation and antibiotic production using genetics and scanning electron microscopy, resulting in authorship on a peer-reviewed manuscript. As a Ph.D. candidate in the laboratory of Dr. Rudolf Jaenisch at the Massachusetts Institute of Technology, I worked on several of projects which resulted in authorship of peer-reviewed manuscripts, related to epigenetics, factor- mediated reprogramming, and transdifferentiation. In my main thesis work, I used genomics, genetics, molecular biology, and tissue culture techniques to study cell fate transitions in the development of the mammalian nervous system, resulting in a first-author publication (Lodato et al., PLoS Genetics, 2013, PMID 23437007). In the first part of my postdoctoral fellowship, in the lab of Dr. Christopher Walsh of Boston Children’s Hospital and Harvard Medical School, I built upon all these previous experiences, in particular in using genomics to understand principles of neurobiology, to study somatic mutations in the normal human brain using cutting-edge single-cell whole genome sequencing (WGS) technologies, resulting in a first-author manuscript (Lodato et al., Science, 2015, PMID 26430121). This paper was the first published work to compare the whole-genome rates and characteristics of somatic mutations across normal human individuals, and the expertise I gained developing that technology has prepared me thoroughly to now compare the rates and characteristics of somatic mutations between age groups and in human age-related diseases. The K99/R00 Pathway to Independence Award is the perfect mechanism to help propel me to my scientific and career goals. Under this award, I will 1) Execute a research plan to uncover the role of somatic mutation in aging and in age-related diseases 2) Gain scientific skills and refine my understanding of key concepts in the fields of aging and bioinformatics 3) Undertake career development activities under the supervision of my mentor, co-mentor, and other faculty advisors committed to my successful transition from postdoctoral fellow to independent investigator. Environment. The proposed Research and Training plans will take place in the laboratory of Christopher A. Walsh, M.D., Ph.D., within Boston Children’s Hospital (BCH) and Harvard Medical School (HMS). These institutions comprise a strong, well-established research community, and are committed to the success of the aims and goals proposed in this application. Dr. Walsh has a strong track record of training postdoctoral fellows, many of his former trainees now holding tenured or tenure-track faculty positions at academic institutions. This success stems from the rigorous and supportive environment in the lab, including frequent lab meetings and one-on-one interactions with Dr. Walsh. Scientifically, the lab is supplied with virtually all reagents and equipment needed for the proposed research, and as a part of the greater BCH and HMS community any additional equipment are available within walking distance. The BCH/HMS community is also an asset to the career development aspects of this proposal, providing easy access to potential collaborators and frequent seminars hosting internal and external speakers. As part of this community, and I have secured a co-mentor (Dr. Peter Park, HMS), and two additional faculty advisors (Drs. Bruce Yanker, HMS, and Emanuela Gussoni, BCH/HMS), all committed to my success. Finally, the BCH/HMS environment provides myriad formal career development seminars and workshops, which I will take part in to aid my transition to independence. Research. Advanced age is a major risk factor for human diseases in all organ systems, yet how the phenomenon of aging affects such a wide spectrum of tissues is unknown. All tissues rely on the integrity of the genome to function properly, and one attractive and long-standing hypothesis is that the gradual accumulation of DNA damage might be causal in aging. Proof of this notion has remained elusive in the brain, since standard DNA-sequencing experiments are ill-suited to detect somatic mutations which might mark only a few cells, or even a single cell, in a sample comprised of millions. I have pioneered the use of single-cell, whole-genome sequencing technology to compare rates, characteristics, and consequences of somatic mutations across human brains, and will use this technology as a part of this award to achieve three Aims: 1) Determine whether aging is associated with an accumulation of mutations in the human brain and define patterns of somatic mutation during aging, 2) Single-cell sequencing in progeroid diseases associated with DNA-damage response, and 3) Examination of the somatic mutation rate in Alzheimer’s disease. Thus, this proposal represents a comprehensive body of work that will elucidate the role of somatic mutation in during aging, in accelerated aging, and in age-associated disease in the human brain.