Understanding the Mechanisms and Consequences of Basement Membrane Aging in Vivo

了解体内基底膜老化的机制和后果

• 批准号: 10465010
• 负责人: William David Ramos-Lewis
• 金额: $ 6.76万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10465010
• 关键词: Adult; Affect; Age; Aging; Animal Model; Animals; Atlases; Atomic Force Microscopy; Automobile Driving; Basement membrane; Blood flow; Caenorhabditis elegans; Cells; Collagen; Collagen Type IV; Complex; Defect; Deposition; Deterioration; Distal; Dystroglycan; Embryo; Epidermis; Excision; Extracellular Matrix; Fertility; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Foundations; Genes; Goals; Gonadal structure; Growth Factor; Health; Hearing; Image; Individual; Integrins; Laminin; Link; Longevity; Maintenance; Measurement; Measures; Mechanics; Mediating; Membrane; Membrane Proteins; Modeling; Muscle; Muscle function; Nerve; Nidogen; Organ; Ovulation; Pathology; Peptide Hydrolases; Pharyngeal structure; Photobleaching; Physiological; Process; Proteins; Pump; Quantitative Microscopy; RNA Interference; Recovery; Regulation; Research; Role; Signal Transduction; Structure; System; Testing; Thick; Thinness; Time; Tissues; Transmission Electron Microscopy; Vertebrates; Vision; Visualization; Work; age related; animal tissue; collagen scaffold; crosslink; experimental study; extracellular; feeding; fluorophore; functional decline; genome editing; germline stem cells; human disease; in vivo; insight; knock-down; mechanical signal; membrane assembly; mutant; oocyte quality; overexpression; perlecan; pharynx muscle; prevent; progenitor; screening; senescence; stem cell division; stem cell niche; stem cells; tool

Project Summary Basement membranes (BMs) are thin, dense, specialized extracellular matrices built on laminin and type IV collagen scaffoldings that underlie and surround most animal tissues. BMs harbor over 100 proteins and provide mechanical, barrier, and signaling support for tissues. Defects in BM assembly and regulation cause embryonic lethality and are linked to numerous human diseases. Aging BMs accumulate type IV collagen and progressively thicken, which is associated with declining tissue function, such as reduced blood flow, vision, hearing, and stem cell renewal. The mechanism(s) driving collagen accumulation and BM thickening during aging, however, are unclear. Furthermore, it has not yet been possible to establish whether and how collagen accumulation causes tissue decline. A key gap in the understanding of BM aging is a lack of animal models that allow visualization of individual BM component accumulation and turnover during aging, as well as the ability to manipulate the levels of BM components and establish their effects on tissue function. The overall objective of this proposal is to develop C. elegans as a powerful new model to elucidate mechanisms of BM aging and its consequences on tissue function. C. elegans offers unique advantages for studying BM aging—a short lifespan (~two weeks), tissue decline during aging is well characterized, all tissues are accessible to live imaging, and facile conditional gene knockdown and overexpression approaches. In addition, most BM components have been endogenously tagged with the genetically encoded fluorophore mNeonGreen (mNG) (~60 BM genes) and core components with mEos2 (photoconvertible), allowing for comprehensive examination of BM component levels and turnover of key components. Preliminary studies have revealed that, similar to vertebrates, collagen IV accumulates dramatically in BMs (as much as 9-fold) on multiple tissues as C. elegans age. Additionally, preventing collagen accumulation via RNAi slows the decline of the germline stem cell niche. To determine the mechanisms and consequences of BM aging, the following specific aims will be pursued: (1) using fluorescent recovery after photobleaching (FRAP), photoconversion, and screening of collagen regulators, the mechanism(s) of collagen IV accumulation in aging BMs will be determined, (2) using RNAi to deplete collagen and examining markers of tissue health and decline, the role of age-dependent BM collagen IV accumulation in stem cell renewal, oocyte quality, ovulation, fertility, and muscle decline will be established, and (3) using a comprehensive toolkit of endogenously tagged BM components, a BM aging atlas will be generated that reveals how BM components change in abundance on all major tissues during aging. The proposed research is significant, as it will establish a new model to study BM aging, elucidate how BM collagen IV accumulates during aging and its effects on tissue decline, and create a comprehensive atlas of BM aging that will reveal additional components with age-related changes that will drive future research on BM regulation, aging, and tissue health.

项目摘要 地下膜（BMS）是薄的，密集的，专门的细胞外矩阵，建立在层粘连蛋白和IV型上 胶原蛋白脚手架是大多数动物组织的基础。 BMS港口超过100种蛋白质， 为组织提供机械，障碍和信号支持。 BM组装和调节原因的缺陷 胚胎致死性，与许多人类疾病有关。老化BMS积累IV型胶原蛋白和 逐渐变厚，这与组织功能下降有关，例如血流降低，视力， 听力和干细胞更新。驱动胶原蛋白积累和BM增厚的机制 但是，衰老尚不清楚。此外，尚无可能确定是否以及如何胶原蛋白 积累会导致组织下降。理解BM衰老的关键差距是缺乏动物模型 这允许在衰老期间可视化单个BM组件的积累和营业额，以及 能够操纵BM组件的水平并确定其对组织功能的影响。总体 该提议的目的是开发秀丽隐杆线虫作为阐明BM机制的强大新模型 衰老及其对组织功能的后果。秀丽隐杆线虫为研究BM衰老提供了独特的优势 - 寿命短（〜两周），衰老期间的组织下降的特征是所有时间都可以使用 成像，有条件的基因敲低和过表达方法。此外，大多数BM 组件已用一般编码的荧光团绿色（MNG）进行了内源标记 （〜60 bm基因）和具有MEOS2（光转换）的核心成分，允许全面 检查BM组件水平和关键组件的周转。初步研究表明， 与脊椎动物类似，胶原蛋白IV在多个时机上在BMS（多达9倍）中显着积累 秀丽隐杆线年的年龄。另外，防止通过RNAi积累胶原蛋白会减慢种系的下降 干细胞生态位。为了确定BM衰老的机制和后果，以下特定目的 将被追捕：（1）在光漂白后使用荧光恢复（FRAP），光转换和筛选 在胶原蛋白调节剂中，将确定BMS胶原蛋白IV积累的机理，（2） 使用RNAi复制胶原蛋白并检查组织健康和衰落的标志物，依赖年龄的作用 BM胶原蛋白IV在干细胞更新，卵母细胞质量，排卵，生育能力和肌肉下降中的积累将是 建立，（3）使用内源标记的BM组件的全面工具包，BM老化Atlas 将生成揭示BM成分在衰老过程中所有主要组织中抽象的变化。 拟议的研究非常重要，因为它将建立一个新的模型来研究BM衰老，阐明BM的方式 胶原蛋白IV在衰老期间积累及其对组织下降的影响，并创建一个全面的地图集 BM衰老将揭示与年龄相关的更改的其他组成部分，这将推动BM的未来研究 调节，衰老和组织健康。