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

描述(申请人提供)：Lowe OculoCerebroRenal综合征(LOCR)是一种罕见的X连锁先天性白内障、智力低下、行为异常和肾小管病变。这种疾病是由OCRL基因功能丧失突变引起的，OCRL基因编码OCRL，一种位于反高尔基网络的磷脂酰肌醇(4，5)P2 5-磷酸酶，内体，质膜褶皱和笼蛋白包裹的凹坑。LOCR的肾小管异常，特别是低分子(LMW)蛋白尿和氨基酸酸尿，与Dent病的肾小管异常重叠，Dent病只影响肾脏。OCRL基因突变约占Dent病患者的20%。已有研究表明，LOCR中磷脂酰肌醇(4，5)P2代谢的紊乱导致受体内吞功能缺陷，如顶端转运或回收megalin，这是一种负责在肾脏近端小管重新吸收LMW蛋白的蛋白质。中枢神经系统异常可能是由于某些中枢神经系统受体在循环过程中出现类似的缺陷而引起的。由于缺乏小鼠模型，这些假说的验证受到阻碍，因为OCRL(尿液中OCRL的同源物)基因敲除的小鼠没有可检测到的表型。我们已经证明，Ocr1基因敲除小鼠的肾脏表型缺失是由于另一种由小鼠Inpp5b和小鼠INPP5B编码的磷脂酰肌醇(4，5)P2 5-磷酸酶Inpp5b完全补偿所致。Inpp5b突变的小鼠只有轻微的表型，但Ocr1和Inpp5b的突变都会导致早期胚胎死亡，这表明功能重叠。INPP5B和Inpp5b在转录和剪接方面有明显的差异，这可以解释为什么INPP5B只能部分补偿人OCRL的丢失，而小鼠Inpp5b可以完全补偿。我们创造了同时缺失小鼠Ocr1和Inpp5b的转基因小鼠，以及含有INPP5B的细菌人工染色体(BAC)的半合子转基因小鼠，发现它们生长延迟，并出现LOCR和Dent病中出现的近端肾小管缺陷。同时缺失小鼠Ocr1和Inpp5b以及INPP5B-BAC插入纯合的小鼠生长更正常，分泌的LMW蛋白要少得多。我们的结论是，INPP5b和Inpp5b在小鼠体内补偿Ocr1丢失的能力存在质的和量的差异。在目前的应用中，我们建议从这些品系的小鼠中培养近端肾小管细胞，并将INPP5B酶的活性与磷脂酰肌醇(4，5)P2的胞下水平以及低分子蛋白质和氨基酸在肾脏的转运改变相关联。我们还将利用小鼠胚胎干细胞中的重组来解剖和鉴定INPP5b和Inpp5b之间的差异，这些差异是导致Ocr1缺乏症人类和小鼠表型差异的原因。最后，在完成了对C57BL/6的10代回交后，我们将通过测试运动功能、焦虑、学习、记忆和脑神经病理检查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.