LIPASE AND CATHESPIN ABNORMALITIES IN BATTEN DISEASE

本提案的长期目标是了解 三种主要形式（婴儿，晚期婴儿， 和青少年）神经元蜡样质脂褐质沉积症（NCL）或Batten病。 这将允许NCL的精确诊断，杂合子的检测， 并促进新形式的治疗， 神经退行性疾病，导致视网膜变性和丧失 所有的神经功能。 我们将检验一个假设，即溶酶体缺乏 磷脂酶A1（PLA 1）可导致生化和病理 在Batten病中观察到的变化。 为了实现这一目标，我们将首先 从人体组织中纯化PLA 1，并确定 最佳活性和底物特异性。 我们将确定PLA 1 从患有Batten病的患者制备的组织匀浆中的活性 来验证我们的初始数据，这些数据显示出明显的缺陷 我们将 测定来自专性NCL载体的永生化淋巴细胞的活性， 通过显示50%的正常活性来验证PLA 1是主要缺陷 在这些航母上。 然后，我们将制备针对PLA 1的多克隆抗体， 用它来研究合成，加工和亚细胞定位 正常细胞与突变细胞中的PLA 1。 由于动物模型在设计治疗方法中的重要性， 对于贮藏性疾病，我们将尝试确认PLA 1缺乏， 使用NCL的英国塞特犬，并使用来自专性载体的组织， 设计可靠的载波测试。 最后，为了了解NCL的发病机制，我们 将研究溶酶体PLA 1缺乏如何引起 “功能性蛋白酶缺乏症”，主要涉及中枢系统 视网膜的神经元和色素上皮细胞。 我们将尝试 用提出的病原体在体外重现疾病 4-羟基壬烯醛，研究2例明显原发性组织蛋白酶H患者 缺乏和临床症状类似NCL，并准备 [125 I]标记的视紫红质和NCL中推定的主要储存肽 (the线粒体ATP合酶的C-亚基）用于体外测定 NCL组织。

The long-term objective of this proposal is to understand the biochemical basis of the three major forms (infantile, late infantile, and juvenile) of neuronal ceroid lipofuscinosis (NCL) or Batten disease. This will permit precise diagnosis of NCL, detection of heterozygotes, and facilitate new forms of therapy for this devastating neurodegenerative disease which causes retinal degeneration and loss of all neurofunctions in 3000 children each year. We will test a hypothesis that a deficiency of lysosomal phospholipase A1 (PLA1) can lead to the biochemical and pathological changes observed in Batten disease. To achieve this, we will first purify the PLA1 from human tissue and determine the conditions for optimum activity and substrate specificity. We will determine PLA1 activity in tissue homogenates prepared from patients with Batten disease to verify our initial data which suggests a marked deficiency. We will assay activity in immortalized lymphocytes from obligate NCL carriers to verify that PLA1 is the primary defect by showing 50% of normal activity in these carriers. We will then prepare polyclonal antibody to PLA1 and use it to study synthesis, processing, and subcellular localization of PLA1 in normal versus mutant cells. Because of the importance of animal models in devising therapy for storage diseases, we will attempt to confirm the PLA1 deficiency in English Setters with NCL, and use tissue from the obligate carriers to devise a reliable carrier test. Finally, in order to understand the pathogenesis of NCL, we will investigate how a lysosomal PLA1 deficiency can give rise to a "functional protease deficiency", involving primarily central system neurons and pigmentary epithelial cells of the retina. We will attempt to recreate the disease in vitro with the proposed pathogenic agent 4-hydroxynonenal, study two patients with apparent primary cathepsin H deficiency and clinical symptoms resembling NCL, and prepare [125I]labeled rhodopsin, and the putative major storage peptide in NCL (the C-subunit of mitochondrial ATP synthase) for use in in vitro assays with NCL tissue.