LIPASE AND CATHESPIN ABNORMALITIES IN BATTEN DISEASE

这项建议的长期目标是了解 三种主要形式的生化基础(婴儿、婴儿晚期、 和青少年)神经性蜡样脂褐素沉着症(NCL)或巴顿病。 这将使NCL的准确诊断、杂合子的检测、 并促进对这种毁灭性疾病的新形式的治疗 一种神经退行性疾病，导致视网膜变性和视神经功能丧失。 每年3000名儿童的所有神经功能。 我们将检验一种假设，即溶酶体缺乏 磷脂酶A1(PLA1)可导致生化和病理改变。 观察到巴顿病的变化。为了实现这一目标，我们将首先 从人体组织中提纯PLA1并确定提取条件 最佳活性和底物专一性。我们将确定PLA1 巴顿病患者组织匀浆的活性 来验证我们最初的数据，这表明我们有一个明显的缺陷。我们会 NCL携带者永生化淋巴细胞的检测活性 通过显示50%的正常活动来验证PLA1是否为主要缺陷 在这些航母上。然后我们将制备针对PLA1的多克隆抗体和 用它来研究合成、加工和亚细胞定位 正常细胞和突变细胞中的PLA1。 因为动物模型在设计治疗方法中的重要性 对于储藏疾病，我们将尝试确认PLA1缺乏 使用NCL的英语二传手，并使用专属携带者的组织来 设计一种可靠的载波测试。 最后，为了了解NCL的发病机制，我们 将研究溶酶体PLA1缺陷是如何引起 “功能性蛋白酶缺乏症”，主要累及中枢系统 视网膜的神经元和色素上皮细胞。我们将尝试 用所提出的病原体在体外重建疾病 4-羟基壬烯醛，研究两名明显患有原发性组织蛋白酶H的患者 缺乏与NCL相似的临床症状，并准备 [125I]标记视紫红质，以及NCL中可能的主要储存肽 (线粒体三磷酸腺苷合成酶的C亚基)用于体外分析 用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.