Combined BAC Transgenic and Knock-Out Mouse Model of Lowe Syndrome Nephropathy

Lowe 综合征肾病的 BAC 转基因和敲除小鼠模型相结合

• 批准号: 8236723
• 负责人: ROBERT L NUSSBAUM
• 金额: $ 33.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8236723
• 关键词: Accounting; Address; Affect; American Society of Hematology; Amino Acids; Animals; Anxiety; Apical; Backcrossings; Bacterial Artificial Chromosomes; Behavioral; Binding; Brain; Cataract; Cell membrane; Cells; Chloride Channels; Clathrin; Complex; Congenic Mice; Defect; Disease; Embryo; Endocytosis; Endosomes; Enzymes; Epitopes; Exons; Financial compensation; Functional disorder; Generations; Genes; Genetic Recombination; Genetic Transcription; Germ Cells; Golgi Apparatus; Growth; Homologous Gene; Human; Human Genome; Immunoprecipitation; Kidney; Kidney Diseases; Knock-out; Knockout Mice; LDL-Receptor Related Protein 2; Learning; Link; Mediating; Memory; Memory impairment; Mental Retardation; Metabolic Pathway; Metabolism; Molecular Weight; Motor; Mouse Strains; Mus; Mutant Strains Mice; Mutation; Neuraxis; Neurologic; Oculocerebrorenal Syndrome; PH Domain; Patients; Peptides; Phenotype; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipids; Phosphoric Monoester Hydrolases; Proteins; Proteinuria; Proximal Kidney Tubules; RNA Splicing; Rare Diseases; Recycling; Research Proposals; Structure; Testing; Transgenic Mice; Transgenic Organisms; Tubular formation; Urine; base; coated pit; cohort; congenital cataract; disease-causing mutation; embryo tissue; embryonic stem cell; enzyme activity; homologous recombination; human tissue; insight; lens; loss of function mutation; motor deficit; mouse genome; mouse model; promoter; receptor; rho; stem; trafficking; trans-Golgi Network; yeast two hybrid system

DESCRIPTION (provided by applicant): The Lowe OculoCerebroRenal syndrome of (LOCR) is a rare X-linked disorder of congenital cataracts, mental retardation, behavioral abnormalities, and renal tubulopathy. The disease is caused by loss-of-function mutations in the OCRL gene, which encodes Ocrl, a phosphatidylinositol(4,5)P2 5-phosphatase located in the trans-Golgi network, endosomes, plasma membrane ruffles, and clathrin-coated pits. The renal tubular abnormalities of LOCR, particularly low molecular weight (LMW) proteinuria and aminoaciduria, overlap with those of Dent disease, a disorder that affects only the kidney. Mutations in OCRL account for ~20% of Dent disease patients. It has been proposed that derangements in phosphatidylinositol(4,5)P2 metabolism in LOCR causes defective receptor endocytosis, such as apical trafficking or recycling of megalin, a protein responsible for reabsorbing LMW proteins in the kidney proximal tubule. The CNS abnormalities could arise by analogous defects in recycling of certain CNS receptors. Testing these hypotheses is hampered by the lack of a mouse model because mice with a knock-out of Ocrl, the urine homolog of OCRL, have no detectable phenotype. We have shown that the lack of renal phenotype in Ocrl-knockout mice is due to complete compensation by another phosphatidylinositol(4,5)P2 5-phosphatase, Inpp5b, encoded by murine Inpp5b and mouse INPP5B. Mice with a mutation in Inpp5b have only a mild phenotype but mutation of both Ocrl and Inpp5b causes early embryonic lethality, indicating functional overlap. INPP5B and Inpp5b have clear differences in transcription and splicing which could explain why INPP5B only partially compensates for loss of human OCRL while mouse Inpp5b completely compensates. We created transgenic mice deleted for both murine Ocrl and Inpp5b and hemizygous for a bacterial artificial chromosome (BAC) containing INPP5B, and found they have delayed growth and the proximal renal tubular defects seen in LOCR and Dent disease. Mice deleted for both murine Ocrl and Inpp5b and homozygous for the INPP5B-BAC insertion grow more normally and excrete much less LMW protein. We conclude that there is both a qualitative and quantitative difference in the ability of INPP5B and Inpp5b to compensate for loss of Ocrl in mice. In this current application, we propose to culture proximal tubular cells from these strains of mice and correlate INPP5B enzyme activity with subcelular levels of phosphatidylinositol(4,5)P2 and altered renal transport of low molecular weight proteins and amino acids. We will also use recombination in mouse embryonic stem cells to dissect and identify the differences between INPP5B and Inpp5b responsible for the difference in phenotype between Ocrl-deficient humans and mice. Finally, after completing a 10-generation backcross onto C57Bl/6, we will examine the mice for abnormalities in central nervous system by testing motor function, anxiety, learning, memory and by examining the brains neuropathologically. PUBLIC HEALTH RELEVANCE: Lowe syndrome is a rare disorder characterized by congenital lens cataracts, mental retardation, behavioral abnormalities, and kidney dysfunction caused by mutations in a gene, OCRL, which encodes an enzyme called Ocrl. Ocrl is part of a very complex metabolic pathway and is involved in the breakdown of phosphatidylinositol phosphate. We have shown that mice with mutations in the mouse version of OCRL have no detectable disease because of complete compensation by another mouse gene, caled Inpp5b, while the human version of this same gene, INPP5B, can only partially compensate for deficiency Ocrl in humans, thus allowing the renal disease to occur. We have made mice lacking both mouse Ocrl and mouse Inpp5b but expressing human INPP5B and have found they mimic the renal disease seen in Lowe syndrome. We are proposing to characterize kidney and brain abnormalities in these mice as well as determine why INPP5B and Inpp5b are different in how completely they compensate for loss of Ocrl in humans versus mice. We expect to gain insight into the metabolic pathways served by Ocrl that are important in causing the renal tubular defects and CNS abnormalities seen in Lowe syndrome.

描述（由申请人提供）：Lowe 眼脑肾综合征 (LOCR) 是一种罕见的 X 连锁疾病，包括先天性白内障、智力低下、行为异常和肾小管病。该疾病是由 OCRL 基因的功能缺失突变引起的，该基因编码 Ocrl，一种位于反高尔基体网络、内体、质膜皱褶和网格蛋白包被小凹中的磷脂酰肌醇 (4,5)P2 5-磷酸酶。 LOCR 的肾小管异常，特别是低分子量 (LMW) 蛋白尿和氨基酸尿，与 Dent 病（一种仅影响肾脏的疾病）的肾小管异常重叠。 OCRL 突变约占 Dent 病患者的 20%。有人提出，LOCR 中磷脂酰肌醇 (4,5)P2 代谢的紊乱会导致受体内吞作用缺陷，例如巨蛋白的顶端运输或再循环，巨蛋白是一种负责在肾近曲小管中重吸收 LMW 蛋白的蛋白。中枢神经系统异常可能是由于某些中枢神经系统受体再循环过程中的类似缺陷引起的。由于缺乏小鼠模型，测试这些假设受到阻碍，因为敲除 Ocrl（OCRL 的尿液同源物）的小鼠没有可检测的表型。 我们已经证明，Ocrl 敲除小鼠中肾脏表型的缺乏是由于另一种磷脂酰肌醇（4,5）P2 5-磷酸酶 Inpp5b（由小鼠 Inpp5b 和小鼠 INPP5B 编码）的完全补偿所致。 Inpp5b 突变的小鼠仅具有轻微的表型，但 Ocrl 和 Inpp5b 突变会导致早期胚胎致死，表明功能重叠。 INPP5B 和 Inpp5b 在转录和剪接方面具有明显差异，这可以解释为什么 INPP5B 只能部分补偿人类 OCRL 的损失，而小鼠 Inpp5b 则完全补偿。我们创建了小鼠 Ocrl 和 Inpp5b 缺失以及含有 INPP5B 的细菌人工染色体 (BAC) 半合子的转基因小鼠，发现它们具有生长延迟以及 LOCR 和 Dent 病中所见的近端肾小管缺陷。小鼠 Ocrl 和 Inpp5b 缺失以及 INPP5B-BAC 插入纯合的小鼠生长更正常，分泌的 LMW 蛋白也少得多。我们得出的结论是，INPP5B 和 Inpp5b 补偿小鼠 Ocrl 缺失的能力存在定性和定量差异。 在当前的应用中，我们建议培养来自这些小鼠品系的近端肾小管细胞，并将 INPP5B 酶活性与磷脂酰肌醇（4,5）P2 的亚细胞水​​平以及低分子量蛋白质和氨基酸的肾脏转运改变相关联。我们还将利用小鼠胚胎干细胞中的重组来剖析和鉴定 INPP5B 和 Inpp5b 之间的差异，这些差异导致 Ocrl 缺陷的人类和小鼠之间的表型差异。最后，在完成与 C57Bl/6 的 10 代回交后，我们将通过测试运动功能、焦虑、学习、记忆以及检查大脑神经病理学来检查小鼠的中枢神经系统异常。 公众健康相关性：Lowe 综合征是一种罕见疾病，其特征是先天性晶状体白内障、智力低下、行为异常和肾功能障碍，由 OCRL 基因突变引起，该基因编码一种名为 Ocrl 的酶。 Ocrl 是非常复杂的代谢途径的一部分，参与磷酸磷脂酰肌醇的分解。我们已经证明，小鼠版本的 OCRL 发生突变的小鼠没有可检测到的疾病，因为另一种小鼠基因 Inpp5b 可以完全补偿，而同一基因的人类版本 INPP5B 只能部分补偿人类中的 Ocrl 缺陷，从而导致肾脏疾病的发生。我们制作了同时缺乏小鼠 Ocrl 和小鼠 Inpp5b 但表达人类 INPP5B 的小鼠，并发现它们模仿了 Lowe 综合征中所见的肾脏疾病。我们提议表征这些小鼠的肾脏和大脑异常，并确定为什么 INPP5B 和 Inpp5b 在补偿人类 Ocrl 缺失的完全程度方面与小鼠不同。我们希望深入了解 Ocrl 所服务的代谢途径，这些途径对于导致 Lowe 综合征中出现的肾小管缺陷和中枢神经系统异常非常重要。