Understanding the Mechanisms and Consequences of Basement Membrane Aging in Vivo

项目摘要 基底膜（BM）是薄的、致密的、特化的细胞外基质，其建立在层粘连蛋白和IV型胶原上。 大部分动物组织的底层和周围的胶原支架。BM含有超过100种蛋白质， 为组织提供机械、屏障和信号支持。BM装配和调节缺陷导致 胚胎致死，并与许多人类疾病有关。老化的BM积累IV型胶原蛋白， 逐渐恶化，这与组织功能下降有关，如血流减少，视力， 听力和干细胞再生治疗期间驱动胶原蛋白积聚和BM增厚的机制 然而，衰老还不清楚。此外，还不可能确定胶原蛋白是否以及如何 积累导致组织衰退。对BM衰老的理解的一个关键差距是缺乏动物模型 其允许在老化过程中可视化单个BM组分的积累和周转，以及 操纵BM组分水平并确定其对组织功能的影响的能力。整体 该提案的目的是开发C。elegans作为一个强大的新模型，以阐明BM的机制 衰老及其对组织功能的影响。C.秀丽线虫为研究BM老化提供了独特的优势， 寿命短（约两周），衰老期间组织衰退的特征很好，所有组织都可以存活 成像和简易的条件性基因敲低和过表达方法。此外，大多数BM 已经用遗传编码的荧光团mNeonGreen（mNG）内源性标记了组分 （约60个BM基因）和核心组分与mEos 2（光转化），允许全面的 检查BM组件水平和关键组件的周转。初步研究显示， 与脊椎动物相似，IV型胶原蛋白在多种组织的BM中显著积累（多达9倍）， C. elegans年龄此外，通过RNA干扰防止胶原蛋白积聚可以减缓种系的衰退 干细胞龛为了确定BM老化的机制和后果，以下具体目标 将追求：（1）使用荧光恢复后光漂白（FRAP），光转换，和筛选 胶原调节剂，将确定老化BM中胶原IV积累的机制，（2） 使用RNAi来消耗胶原蛋白，并检查组织健康和衰退的标志物，年龄依赖性 BM IV型胶原蛋白的积累会在干细胞更新、卵母细胞质量、排卵、生育能力和肌肉衰退中 建立，和（3）使用一个全面的工具包内源性标记的BM组件，BM老化图谱 将产生揭示BM组分如何在老化期间在所有主要组织上大量变化的结果。 本研究将建立一种新的研究BM衰老的模型，阐明BM衰老的机制， 胶原蛋白IV在衰老过程中的积累及其对组织衰退的影响，并创建了一个全面的图谱， BM老化将揭示与年龄相关变化的其他成分，这将推动未来对BM的研究 调节、老化和组织健康。

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.