LIPASE AND CATHEPSIN ABNORALITIES IN BATTEN DISEASE

这是对一项申请的更新，其广泛的长期目标是 了解巴顿病的遗传和生化基础 (神经元性胶质细胞减少症；NCL)。我们将特别强调 关于婴儿晚期(NCL2)和少年(NCl3)形式的认识 由于这些疾病是遗传性失明、癫痫的常见形式 和美国的痴呆症。我们将测试NCL导致 不能运输和降解正常的线粒体蛋白( 线粒体三磷酸腺苷合成酶蛋白脂亚单位c-DCcd结合 缩写为多肽)。这种多肽的溶酶体储存，主要是 在神经元、视网膜色素上皮细胞和胰腺中， 假定会引起许多溶酶体常见的临床症状 储藏疾病。我们将从NCL组织中提纯c-肽，标记它 用[14C]DCD或[125I]，用特异性多克隆抗体鉴定， 检查是否有任何异常的翻译后修饰，如三甲基- 赖氨酸残基，以及正常和NCL负荷后的分解代谢 成纤维细胞和淋巴母细胞系。这只会间接地 因此，我们已经通过聚合酶链式反应获得了绵羊C-肽的cdna，并将其拼接到 一种含有巨细胞病毒启动子等且稳定的 该基因在神经母细胞瘤细胞系F-11中过表达。这些和相关的 LINES是CNS的典范。我们还将同时转导正常和NCL 淋巴母细胞系。我们将尝试创建自动荧光 色素储存，然后显示这也会导致二元醇糖分 蓄积、溶酶体磷脂酶A1抑制等生化 NCL的特点。我们将确定泛素途径是否 通常参与c-肽的处理，寻找c-肽的转运 蛋白质的C-肽亲和层析脑提取液及外观 C-肽在脂蛋白复合体中的浮选密度 在1.063-1.21g/ml范围内对氯化铯的梯度 超速离心机。任何突变都会在大量的细胞中进行检测 来自患者的专性杂合子(正常的50% 活动)，并将制定可靠的诊断测试。理解 NCL的缺陷显然将开辟细胞生物学的一个全新领域 处理疏水性多肽的运输和处置。最后， ，检测载体的能力将在打击 这种毁灭性的儿童神经退行性疾病。

This is a renewal of an application whose broad, long-term objectives are to understand the genetic and biochemical basis of Batten disease (Neuronal Ceroid Liofuscinosis; NCL). Particular emphasis will be placed on understanding the late-infantile (NCL2) and juvenile (NCL3) forms of the disease since these are common forms of inherited blindness, seizures and dementia in the U.S. We will test the hypothesis that NCL results from the inability to transport and degrade a normal mitochondrial protein (the DCCD-binding, proteolipid subunit c of mitochondrial ATP synthase (c- peptide for short). The lysosomal storage of this peptide, predominantly in neurons, retinal pigmentary epithelial cells and pancreas, is then hypothesized to give rise to clinical symptoms common to many lysosomal storage diseases. We will purify the c-peptide from NCL tissue, label it with [14C]DCCD or [125I], identify it by specific polyclonal antibody, check for any abnormal post-translational modifications such as trimethyl- lysine residues, and follow its catabolism after loading of normal and NCL fibroblasts and lymphoblastoid cell lines. This will only give indirect evidence so we have obtained cDNA for sheep c-peptide by PCR, spliced into a plasmid vector which contains a cytomegalovirus promoter etc. and stably overexpressed the gene in neuroblastoma cell line F-11. These and related lines are a model for the CNS. We will also transfect both normal and NCL lymphoblastoid cell lines. We will attempt to create autofluorescent pigment storage and then show that this also results in dolichol sugar accumulation, lysosomal phospholipase A1 inhibition.and other biochemical characteristics of NCL. We will determine if the ubiquitin pathway is normally involved in c-peptide disposal, look for a c-peptide transport protein by c-peptide affinity chromatography of brain extracts and look for c-peptide in lipoprotein complexes by their flotation densities in the range of 1.063-1.21g/ml on cesium chloride gradients in the ultracentrifuge. Any mutation will be assayed in the large number of cell lines available from patients, obligate heterozygotes (50% of normal activity) and a reliable diagnostic test will be devised. Understanding the defect in NCL will clearly open up a, whole new area of cell biology dealing with the transport and disposal of hydrophobic peptides. Finally, the, ability to detect carriers will have tremendous value in combatting this devastating neurodegenerative disorder of children.