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）：溶酶体疾病例如IV型粘脂沉积症（MLIV）的治疗策略非常有限。 MLIV 是一种先天性疾病，其特征是眼、中枢神经系统、胃肠道和其他器官的晚期内体和溶酶体过程紊乱。这是一种毁灭性的疾病，通常在生命的第一年出现失明和认知障碍。它是由 MCOLN1 突变引起的，MCOLN1 是编码 mucolipin-1 (ML) 的基因，我们最近建立了该基因来代表由 Ca2+ 瞬时调节的 Ca2+ 渗透性阳离子通道。 此外，我们的工作表明，自然发生的突变 ML 通道仅被细胞内 Ca2+ (Cai) 的增加微弱激活，并表现出其他功能异常。我们对 MLIV 患者的成纤维细胞进行了表征，发现 Ca2+ 信号传导受到干扰，并且细胞定位发生改变的大溶酶体。这些细胞中其他显着的表型改变包括晚期内体/溶酶体 (LEL) 融合缺陷和溶酶体胞吐作用紊乱。这些是与调节 Ca2+ 稳态和脂质代谢的信号通路相关的 Ca2+ 依赖性过程。持续需要针对 MLIV 和其他溶酶体疾病的有效疗法。在最近关于溶酶体贮积症的研究中，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物质的积累。 在初步研究中，我们发现类似的药物与饮食和刺激参与 Ca2+ 稳态和脂质代谢的信号通路的特定药物相结合，可以减少 MLIV 患者细胞中的溶酶体增大并刺激 LEL 融合以及溶酶体胞吐作用。 我们还能够通过基因替换方法挽救相同的表型改变。这项研究的总体目标是描述这些表型改变的特征，并设计新的方法来纠正和挽救它们，使用基因替换以及协同作用的饮食和药物制剂的组合。我们将描述 MLIV 患者细胞中发生的溶酶体形态（目标 1）、离子和代谢调节（目标 2）、膜囊泡融合（目标 3）和胞吐作用（目标 4）的病理改变。根据我们的初步研究，我们还将建立通过基因替换拯救所有这些细胞表型的最佳条件。由于目标 5 中基因替代干预措施的一些局限性，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拯救与 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，作为潜在的新兴疗法，不仅适用于 MLIV，还适用于不同类型的粘脂贮积症和其他溶酶体贮积症。