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

描述（由申请人提供）：Lowe眼脑肾综合征（LOCR）是一种罕见的先天性白内障、智力低下、行为异常和肾小管病变的X连锁疾病。该疾病是由OCRL基因中的功能丧失突变引起的，该基因编码Ocrl，一种位于高尔基体网络、内体、质膜皱褶和网格蛋白包被的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病重叠，Dent病是一种仅影响肾脏的疾病。OCRL突变占Dent病患者的约20%。已经提出LOCR中磷脂酰肌醇（4，5）P2代谢的紊乱导致受体内吞作用缺陷，例如巨蛋白（一种负责在肾近端小管中重吸收LMW蛋白的蛋白质）的顶端运输或再循环。中枢神经系统的异常可能是由于某些中枢神经系统受体的再循环类似缺陷引起的。缺乏小鼠模型阻碍了对这些假设的检验，因为Ocrl（OCRL的尿液同源物）基因敲除的小鼠没有可检测的表型。 我们已经表明，Ocrl基因敲除小鼠中肾表型的缺乏是由于另一种磷脂酰肌醇（4，5）P2 5-磷酸酶Inpp 5 b（由鼠Inpp 5 b和小鼠INPP 5 B编码）的完全补偿。具有Inpp 5 b突变的小鼠仅具有轻度表型，但Ocrl和Inpp 5 b两者的突变导致早期胚胎致死，表明功能重叠。INPP 5 B和Inpp 5 b在转录和剪接方面有明显的差异，这可以解释为什么INPP 5 B只能部分补偿人类OCRL的损失，而小鼠Inpp 5 b完全补偿。我们创建了鼠Ocrl和Inpp 5 b缺失的转基因小鼠和含有INPP 5 B的细菌人工染色体（BAC）的半合子小鼠，并发现它们具有延迟的生长和在LOCR和Dent病中观察到的近端肾小管缺陷。小鼠Ocrl和Inpp 5 b缺失和INPP 5 B-BAC插入纯合的小鼠生长更正常，分泌更少的LMW蛋白。我们得出结论，INPP 5 B和Inpp 5 b补偿小鼠中Ocrl损失的能力存在定性和定量差异。 在本申请中，我们提出培养来自这些品系小鼠的近端肾小管细胞，并将INPP 5 B酶活性与磷脂酰肌醇（4，5）P2的细胞下水平以及低分子量蛋白质和氨基酸的改变的肾转运相关联。我们还将使用小鼠胚胎干细胞中的重组来解剖和鉴定INPP 5 B和Inpp 5 b之间的差异，这些差异导致Ocrl缺陷的人和小鼠之间的表型差异。最后，在完成到C57 B1/6的10代回交后，我们将通过测试运动功能、焦虑、学习、记忆和通过神经病理学检查大脑来检查小鼠中枢神经系统的异常。

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.