Gain-of-function toxicity in alpha-1 antitrypsin deficient type 2 alveolar epithelial cells

α-1 抗胰蛋白酶缺陷型 2 型肺泡上皮细胞的功能获得毒性

• 批准号: 10751760
• 负责人: Carly Merritt
• 金额: $ 5.27万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751760
• 关键词: Accounting; Activities of Daily Living; Acute; Air; Alpha Cell; Anatomy; Apical; Biology; Blood Circulation; CRISPR correction; Cell Line; Cell physiology; Cell surface; Cells; Cellular Stress; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; Data; Development; Distal; Endoplasmic Reticulum; Environment; Environmental Risk Factor; Epithelial Cells; Exposure to; Extrahepatic; Functional disorder; Genes; Genetic Diseases; Genetic Engineering; Genetic Transcription; Goals; Hepatocyte; Histologic; Human; In Vitro; Injury; Innate Immune Response; Leukocyte Elastase; Liquid substance; Liver diseases; Location; Lung; Measures; Mediating; Modeling; Mus; Mutate; Mutation; Pathogenesis; Patients; Peptide Hydrolases; Persons; Play; Pluripotent Stem Cells; Protease Inhibitor; Proteins; Publishing; Pulmonary Emphysema; Regenerative Medicine; Role; Severities; Structure of parenchyma of lung; Supplementation; Surface; Testing; Therapeutic; Touch sensation; Toxic effect; United States; alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency; alveolar epithelium; cell type; cigarette smoke; differentiation protocol; directed differentiation; disease-causing mutation; epithelial stem cell; experience; functional loss; gain of function; gene correction; immune cell infiltrate; in vivo; induced pluripotent stem cell; monocyte; mouse model; novel; proteotoxicity; response; response to injury; single-cell RNA sequencing; stem cells; surfactant; transcriptomics; treatment strategy

Alpha-1 antitrypsin deficiency (AATD) is the sole monogenic cause of emphysema. AATD is caused by a mutation in the SERPINA1 gene, which encodes for alpha-1 antitrypsin (AAT). AAT is produced mainly in hepatocytes, from which it is secreted into the bloodstream to exert its primary function of inhibiting neutrophil elastase in the lungs. The most common disease-causing mutation results in polymerogenic AAT, known as “Z AAT”. Z AAT is trapped in the endoplasmic reticulum of hepatocytes, leading to proteotoxic liver disease and reduced circulating AAT. AATD emphysema has classically been attributed to the protease-antiprotease imbalance resulting from reduced circulating AAT. This hypothesis is complicated by two findings – 1) the supplementation of functional AAT is less effective than expected if emphysema were solely caused by loss of AAT and 2) there is evidence for Z AAT-induced proteotoxicity in extrahepatic cells. Type 2 alveolar epithelial cells (AT2s) are the progenitor cells of the distal lung, renewing both themselves and type 1 alveolar epithelial cells in response to injury. While Z AAT expression has been shown in multiple extrahepatic cell types, and recently published single cell RNA sequencing data have shown high expression of SERPINA1 in AT2s, AAT expression in AT2s remains unexplored. Our preliminary data identifies transcriptional evidence of an unfolded protein response in AT2s from explanted human PiZZ lungs. The goal of this proposal is to elucidate the functional and transcriptional impacts of Z AAT expression in AT2s, both at baseline and in the context of cigarette smoke injury, and to determine whether Z AAT expression in AT2s contributes to the pathogenesis of emphysema. Primary human AT2s are difficult to access pre-mortem. It is likewise challenging to maintain primary human AT2s in vitro, limiting studies of their biology. To solve these challenges, we will utilize the combination of a novel mouse model in Aim 1 and AATD patient-derived induced pluripotent stem cells (iPSCs) in Aim 2. In Aim 1, we will chronically expose mice expressing human Z or “normal” SERPINA1 cDNA in AT2s to cigarette smoke to investigate the accumulation of AAT and activation of the unfolded protein response in AT2s, as well as histologically examine emphysema severity and immune cell infiltration. In Aim 2, we will to generate AT2-like cells (iAT2s) from AATD patient-derived and CRISPR-corrected isogenic control iPSCs and expose them to cigarette smoke. We will determine iAT2 dysfunction and genetic disease signature by measuring AAT protein accumulation and secretion, activation of the unfolded protein response, functional capacity, and global transcriptomic response to injury. Together these Aims will produce significant advances in our understanding of AATD, and suggest the need for novel treatment strategies.

α-1抗胰蛋白酶缺乏(AATD)是肺气肿的唯一单基因致病因素。AATD是由一种 编码α-1抗胰蛋白酶(AAT)的SERPINA1基因突变。AAT主要生产在 肝细胞，从肝细胞分泌到血液中，发挥其抑制中性粒细胞的主要功能 肺中的弹性蛋白酶。最常见的致病突变会导致多基因AAT，也就是众所周知的“Z” AAT“。ZAAT被困在肝细胞的内质网中，导致蛋白毒性肝病和 降低了循环AAT。AATD肺气肿被经典地归因于蛋白水解酶-抗蛋白酶。 循环AAT减少造成的失衡。这一假设因两个发现而变得复杂-1) 如果肺气肿完全是由于肺气肿缺乏引起的，补充功能性AAT的效果不如预期 2)有证据表明Z-AAT对肝外细胞有蛋白毒性作用。2型肺泡上皮细胞 细胞(AT2)是远端肺的前体细胞，既能更新自身，也能更新1型肺泡上皮 细胞对损伤的反应。而Z-AAT在多种肝外细胞类型中都有表达，并且 最近发表的单细胞RNA测序数据显示，SERPINA1在AT2s、AAT中高表达 在AT2中的表达仍未被研究。我们的初步数据确定了一种未展开的 移植的人披萨肺中AT2的蛋白质反应。这项提议的目的是澄清 ZAAT在AT2s中表达的功能和转录影响，无论是在基线还是在 香烟烟雾损伤，并确定Z-AAT在AT2s的表达是否参与了高血压的发病机制。 肺气肿。原始人AT2很难在死前接触到。维护它也同样具有挑战性 原代人类AT2在体外，限制了对其生物学的研究。为了解决这些挑战，我们将利用 AIM-1新小鼠模型与AATD患者来源的诱导多能干细胞(IPSCs)的结合 在目标2中。在目标1中，我们将在AT2s中慢性暴露表达人Z或正常的SERPINA1基因的小鼠 香烟烟雾对大鼠脑内AAT积聚和未折叠蛋白反应的影响 AT2s，以及组织学检查肺气肿的严重程度和免疫细胞的渗透。在目标2中，我们将 从AATD患者来源和CRISPR校正的等基因对照IPSCs中生成AT2样细胞(IAT2) 让他们暴露在香烟烟雾中。我们将通过以下方式确定iAT2功能障碍和遗传病特征 测量AAT蛋白的积累和分泌，激活未折叠的蛋白反应，功能 能力，以及对损伤的全球转录反应。这些目标加在一起将在以下方面取得重大进展 我们对AATD的理解，并建议需要新的治疗策略。