Elucidation and rescue of phenotypic alterations in MLIV

MLIV 表型改变的阐明和挽救

• 批准号: 7066658
• 负责人: PETER M VASSILEV
• 金额: $ 37.76万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7066658
• 关键词: Xenopus oocyte; autosomal recessive trait; biotechnology; clinical research; combination therapy; diet therapy; drug screening /evaluation; exocytosis; fluorescent dye /probe; gene therapy; genetic polymorphism; human tissue; inborn lipid storage disorder; inborn lysosomal enzyme disorder; metabolism disorder chemotherapy; phenotype; point mutation; site directed mutagenesis; small interfering RNA; therapy design /development

DESCRIPTION (provided by applicant): The therapeutic strategies for lysosomal diseases such as mucolipidosis type IV (MLIV) are very limited. MLIV is an inborn disease characterized by disturbances in late endosomal and lysosomal processes in eye, CNS, gastrointestinal tract and other organs. It is a devastating disorder that usually presents during the first year of life with blindness and cognitive impairment. It is caused by mutations in MCOLN1, the gene encoding mucolipin-1 (ML), which we have recently established to represent a Ca2+ -permeable cation channel that is transiently modulated by Ca2+. In addition, our work has shown that naturally occurring mutant ML channels are only weakly activated by increases in intracellular Ca2+ (Cai) and show other functional abnormalities. We have characterized fibroblasts from MLIV patients and found disturbed Ca2+ signaling and large lysosomes with altered cellular localization. Other significant phenotypic alterations in these cells include defective late endosome/lysosome (LEL) fusion and disturbance of lysosomal exocytosis. These are Ca2+ dependent processes related to signaling pathways regulating Ca2+ homeostasis and lipid metabolism. There is a continued need for effective therapies for MLIV and other lysosomal disorders. In recent studies on lysosomal storage disorders the accumulation of mucoid and lipid materials in lysosomes was reduced using activators of lysosomal enzymes and other catabolic processes. In preliminary studies we found that similar agents in combination with dietary and defined pharmacological agents stimulating signaling pathways involved in Ca2+ homeostasis and lipid metabolism reduced the lysosomal enlargements and stimulated LEL fusion as well as lysosomal exocytosis in cells derived from patients with MLIV. We were also able to rescue the same phenotypic alterations by gene replacement approaches. The overall goal of this study is to characterize these phenotypic alterations and to design new approaches to correct and rescue them using gene replacement and combinations of synergistically acting dietary and pharmacological agents. We will characterize the pathological alterations in lysosomal morphology (Aim 1), ionic and metabolic regulation (Aim 2), membrane vesicle fusion (Aim 3) and exocytosis (Aim 4) occurring in the cells of patients with MLIV. Based on our preliminary studies we will also establish the optimal conditions to rescue all of these cellular phenotypes by gene replacement. Due to some of the limitations of the gene replacement interventions in Aim 5 we also propose alternative approaches based on stimulation of compensatory factors that may also contribute to the correction and rescue of each of the cellular phenotypes that are associated with MLIV. In the long term, these studies should help to develop new treatment modalities as potentially emerging therapies not only for MLIV but also for different types of mucolipidoses and other lysosomal storage disorders.

描述（由申请人提供）：溶酶体疾病（如粘膜脂肪性病IV型（MLIV））的治疗策略非常有限。 MLIV是一种天生的疾病，其特征是眼睛，中枢神经系统，胃肠道和其他器官的内体晚期和溶酶体过程中的干扰。这是一种毁灭性疾病，通常在生命的第一年出现失明和认知障碍。它是由MCOLN1的突变引起的，MCOLN1是编码粘脂蛋白-1（ML）的基因，我们最近已经建立了代表Ca2+可渗透阳离子通道，该阳离子通道由Ca2+暂时调节。 此外，我们的工作表明，天然存在的突变ML通道仅通过细胞内Ca2+（CAI）的增加而被薄弱地激活，并显示其他功能异常。我们已经表征了来自MLIV患者的成纤维细胞，发现Ca2+信号传导和大细胞定位的大溶酶体受到干扰。这些细胞中的其他显着表型改变包括晚期内体/溶酶体（LEL）融合和溶酶体胞吐作用的干扰。这些是与调节Ca2+稳态和脂质代谢的信号通路有关的CA2+依赖性过程。继续需要有效治疗MLIV和其他溶酶体疾病。在有关溶酶体储存障碍的最新研究中，使用溶酶体酶和其他分解代谢过程的激活剂减少了溶酶体中粘液和脂质材料的积累。 在初步研究中，我们发现，与饮食和确定的药理学相似的药物相似的药物刺激了参与Ca2+稳态和脂质代谢的信号通路，降低了来自MLIV患者的细胞的溶酶体增大，并刺激了LEL融合型的溶酶体肿瘤。 我们还能够通过基因替代方法来挽救相同的表型改变。这项研究的总体目标是表征这些表型改变，并设计新的方法，以使用基因替代品和协同作用的饮食和药理剂的组合来纠正和营救它们。我们将表征溶酶体形态学（AIM 1），离子和代谢调节（AIM 2），膜囊泡融合（AIM 3）和胞吐作用（AIM 4）发生的病理改变（AIM 4）。基于我们的初步研究，我们还将建立最佳条件，以通过基因替换来挽救所有这些细胞表型。由于AIM 5中基因置换干预措施的某些局限性，我们还提出了基于刺激补偿因子的替代方法，这些方法也可能有助于纠正和营救与MLIV相关的每种细胞表型。从长远来看，这些研究应有助于开发新的治疗方式，不仅是MLIV，而且还针对不同类型的粘脂蛋白和其他溶酶体储存障碍。