HIGH THROUGHPUT SINGLE CELL TRANSCRIPTOMIC APPROACH TO IDENTIFY SUSCEPTIBLE CELL TYPES AND GENE EXPRESSION CHANGES IN HUMAN GLAUCOMA

高通量单细胞转录组学方法鉴定人类青光眼的易感细胞类型和基因表达变化

• 批准号: 10308415
• 负责人: JOSHUA R SANES
• 金额: $ 16.39万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308415
• 关键词: Address; Affect; Age; American; Atlases; Autopsy; Blindness; Brain; Callithrix; Cell Nucleus; Cell Survival; Cells; Characteristics; Clinic; Clinical; Complex; Computer Analysis; Data; Development; Disease; Eye; Foundations; Freezing; Frequencies; Gene Expression; Gene Expression Alteration; Gene Expression Profile; Genes; Genetic; Glaucoma; Homologous Gene; Human; Individual; Investigation; Knowledge; Learning; Macaca; Mammals; Mediating; Methods; Modeling; Molecular; Mus; Neuraxis; Neuronal Injury; Optic Nerve; Patients; Pattern; Persons; Physiologic Intraocular Pressure; Predisposition; Primates; Process; Protocols documentation; Race; Resources; Rest; Retina; Retinal Ganglion Cells; Risk Factors; Rodent Model; Sampling; Small Nuclear RNA; Testing; Tissues; Vision; Visual; base; blind; cell type; differential expression; experience; fovea centralis; ganglion cell; high intraocular pressure; human tissue; improved; insight; macula; modifiable risk; mouse model; nerve damage; non-genetic; nonhuman primate; overexpression; preservation; programs; resilience; selective expression; sex; single-cell RNA sequencing; transcriptome; transcriptome sequencing; transcriptomics; treatment strategy; visual information

ABSTRACT Glaucoma, the leading cause of irreversible blindness worldwide, results from loss of retinal ganglion cells (RGCs), which carry visual information from the eye to the rest of the brain. Current treatment strategies center on lowering intraocular pressure (IOP), the only known modifiable risk factor for glaucoma. However, significant unmet need persists, and a neuroprotective strategy targeting glaucomatous RGCs directly would offer a powerful complementary approach. To date, however, no neuroprotective therapies have successfully entered the clinic. One major obstacle is that little is known about which of many human RGC types are most susceptible, and what molecular changes occur in RGCs prior to their demise. This project uses single cell transcriptomic profiling and integrative computational analysis to address these gaps in our knowledge. Over the past five years, we have helped to develop methods for high throughput single cell RNA sequencing (scRNA-seq) and applied them to retina – first in mice, then in non-human primates, and subsequently in humans and in mouse models of neuronal injury. Most recently, we have implemented the related method of single nucleus RNA-seq (snRNA-seq) so that we can profile tissue obtained post-mortem, frozen and banked. We now propose to obtain and analyze single nucleus transcriptomes of RGCs from well-characterized glaucomatous and normal human retinas. The number of samples must be large, because glaucoma is a heterogeneous disease with diverse genetic and non-genetic risk factors. It is therefore important to study diverse groups, so we can determine whether they converge on common molecular patterns. Specifically, we will profile at least 2000 RGCs from each of 200 human donor eyes, 150 from individuals with verified glaucoma and 50 from age-, sex- and race-matched controls. From the data we obtain, we will (a) determine whether specific RGC types are selectively resilient or vulnerable in glaucoma and (b) identify genes differentially expressed between glaucomatous and normal RGCs of each type. Finally, we will perform similar analysis on a high-IOP mouse model of glaucoma, helping us understand the extent to which diseases processes in humans are accurately modeled in mice. Our results will provide a powerful resource of sufficient power to transform our view of this prevalent, complex and incompletely understood blinding disease.

抽象的 青光眼是全球不可逆失明的主要原因，是由于视网膜丧失而导致的 神经节细胞（RGC）将视觉信息从眼睛到其他大脑的其余部分。 当前的治疗策略降低眼压（IOP），唯一已知的 青光眼的可修改风险因素。但是，重要的未满足需求仍然存在，一个 针对青光眼RGC的神经保护策略直接提供强大的 完全接近。但是，迄今为止，尚无神经保护疗法成功 进入诊所。一个主要障碍是，对许多人类RGC中的哪一个知之甚少 类型最容易受到影响，并且在RGC灭亡之前发生了哪些分子变化。 该项目使用单细胞转录组分析和集成计算分析 根据我们的知识解决这些差距。在过去的五年中，我们帮助发展 高吞吐量单细胞RNA测序（SCRNA-SEQ）的方法，并将其应用于 视网膜 - 首先在老鼠中，然后是非人类隐私，随后在人类和老鼠中 神经元损伤的模型。最近，我们实施了相关的单个方法 核RNA-Seq（SnRNA-Seq），因此我们可以介绍验尸，冷冻和 银行。我们现在建议从RGC中获取和分析RGC的单核转录组 特征良好的青光眼和正常的人视网膜。样本的数量必须是 大，因为青光眼是一种具有潜水遗传和非遗传风险的异质性疾病 因素。因此，重要的是研究潜水小组，因此我们可以确定他们是否 汇聚在常见的分子模式上。具体来说，我们将至少从中介绍2000个RGC 200人眼睛中的每一个，150只来自经过验证的青光眼的人，50个来自年龄的人， 性别和竞赛的控件。从我们获得的数据中，我们将（a）确定是否具体 RGC类型在青光眼中有选择性地弹性或易受伤害，（b）差异鉴定基因 在每种类型的青光眼和正常RGC之间表达。最后，我们将表演 对高素质小鼠模型的类似分析，有助于我们了解 在小鼠中精确建模了人类中的疾病过程。我们的结果将提供 有足够力量的强大资源来改变我们对这种普遍，复杂和的看法 不完全了解盲目疾病。