Lysosomal Diseases

溶酶体疾病

• 批准号: 10008747
• 负责人: Rosa Puertollano-Moro
• 金额: $ 53.67万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10008747
• 关键词: Acids; Acute; Address; Adult; Animal Disease Models; Antigen Presentation; Arginine; Atrophic; Autophagosome; Birth; Bone remodeling; Cardiac; Cardiomyopathies; Cations; Cell Survival; Cell membrane; Cell physiology; Cells; Characteristics; Childhood; Cholesterol Homeostasis; Clinic; Clinical; Collaborations; Cornea; Cytoplasm; Defect; Disease; Down-Regulation; Drug Screening; Embryonic Development; Enzymes; Excision; FRAP1 gene; Functional disorder; Ganglioside Sialidase Deficiency Disease; Generations; Genes; Glucose; Glycogen; Glycogen storage disease type II; Glycosaminoglycans; Hair Cells; Human; Infant; Injections; Ion Channel; Knock-out; Laboratories; Lipids; Lysosomes; Maintenance; Mediating; Membrane; Metabolic Diseases; Modeling; Molecular; Molecular Chaperones; Mus; Muscle; Muscle Cells; Muscle function; Muscular Atrophy; Muscular Dystrophies; Mutation; Myopathy; Optic Atrophy; Organelles; Organism; Pathogenesis; Pathogenicity; Pathology; Patients; Permeability; Pharmacology; Prevalence; Proteins; Recombinants; Residual state; Retinal; Retinal Degeneration; Role; Signal Transduction; Site; Skeletal Muscle; Strabismus; Supplementation; Surface; Testing; Therapeutic; Therapeutic Intervention; Tissues; Translating; Variant; Visual; Wasting Syndrome; Work; Zebrafish; cellular pathology; design; enzyme activity; enzyme replacement therapy; genome editing; heart function; improved; in vivo; insight; interest; knock-down; muscle form; negative affect; neuromuscular; novel; patch clamp; prevent; rare genetic disorder; receptor; repaired; restoration; small molecule; trafficking

Mucolipidosis type IV (MLIV) is an autosomal recessive disorder characterized by acute psychomotor delays, achlorydria, and visual abnormalities including retinal degeneration, corneal clouding, optic atrophy, and strabismus. Lysosomal inclusions are found in most tissues in MLIV patients. The composition of the storage material is heterogeneous and includes lipids and mucopolysaccharides forming characteristic multiconcentric lamellae, as well as soluble, granulated proteins. MLIV is caused by mutations in mucolipin-1 (MCOLN1, also known as TRPML1), an endo-lysosomal cation channel belonging to the transient receptor potential (TRP) superfamily of ion channels. Whole cell patch clamp, as well as recording of native endolysosomal membranes, suggest that MCOLN1 functions as an inwardly (from lumen to cytoplasm) rectifying channel permeable to Ca2+, Na+, K+ and Fe2+/ Mn2+ whose activity is potentiated by low pH. To better understand the pathology of this disease, we used genome editing to knockout the two mcoln1 genes present in Dario rerio (zebrafish). Our model successfully reproduced the retinal and neuromuscular defects observed in MLIV patients, indicating that this model is suitable for studying the disease pathogenesis. Importantly, our model revealed novel insights into the origins and progression of the MLIV pathology, including the contribution of autophagosome accumulation to muscle dystrophy and the role of mcoln1 in embryonic development, hair cell viability and cellular maintenance. The generation of a MLIV model in zebrafish is particularly relevant given the suitability of this organism for large-scale in vivo drug screening, thus providing novel opportunities for therapeutic discovery. Pompe disease, a severe muscle wasting disorder characterized by altered lysosomal function. Profound muscle atrophy is a hallmark of Pompe disease, a rare genetic disorder caused by a deficiency of acid alphaglucosidase (GAA), the enzyme that breaks down glycogen to glucose within lysosomes. Absence of the enzyme leads to a rapidly fatal cardiomyopathy and skeletal muscle myopathy in infants; low levels of residual enzyme activity are associated with childhood and adult onset progressive skeletal muscle myopathy usually without cardiac involvement. Recently, we found dysregulation of mTOR signaling in the diseased muscle cells and focused on the identification of potential sites for therapeutic intervention. Importantly, reactivation of mTOR in the whole muscle of Pompe mice by TSC knockdown or arginine supplementation resulted in the reversal of atrophy and a striking removal of autophagic buildup. The only available therapy for Pompe disease is enzyme replacement therapy (ERT) with human recombinant GAA. This therapy restores cardiac function, but its effect in skeletal muscle is much less robust. The massive autophagic buildup in Pompe skeletal muscle negatively affects the trafficking and lysosomal delivery of the recombinant enzyme. Since we showed that restoration of mTORC1 activity in Pompe skeletal muscle dramatically reduces autophagosome accumulation, we evaluated whether ERT may work more efficiently when autophagic buildup is removed or diminished. For this, we used a dual approach in which restoration of mTORC1 activity by TSC depletion was combined with ERT. Notable, this approach resulted in increased muscle mass and reduced glycogen accumulation, suggesting reversal of the lysosomal pathology. Therefore, we proposed that a combination of TSC-mediated activation of mTOR with ERT may have the potential to address multiple aspects of the disease pathology. An alternative approach to improve treatment of Pompe patients is designing a more efficient recombinant GAA. In collaboration with Amicus Therapeutics, we have tested a novel rhGAA (ATB200) that has substantially higher M6P content, thus improving interaction with the CIM6PR and delivery to target tissues. Injection of ATB200 in a murine Pompe model, together with a small-molecule pharmacological chaperone that prevents loss of activity and denaturalization, significantly reversed intralysosomal glycogen accumulation and autophagic build-up, leading to improved muscle function.

IV 型粘脂沉积症 (MLIV) 是一种常染色体隐性遗传疾病，其特征为急性精神运动迟缓、胃酸缺乏和视觉异常，包括视网膜变性、角膜混浊、视神经萎缩和斜视。 MLIV 患者的大多数组织中均发现溶酶体包涵体。储存材料的成分是异质的，包括形成特征性多同心片层的脂质和粘多糖，以及可溶性颗粒状蛋白质。 MLIV 是由 mucolipin-1（MCOLN1，也称为 TRPML1）突变引起的，粘蛋白-1 是一种内溶酶体阳离子通道，属于离子通道瞬时受体电位 (TRP) 超家族。全细胞膜片钳以及天然内溶酶体膜的记录表明，MCOLN1 充当可渗透 Ca2+、Na+、K+ 和 Fe2+/Mn2+ 的内部（从腔到细胞质）整流通道，其活性因低 pH 值而增强。为了更好地了解这种疾病的病理学，我们使用基因组编辑来敲除 Dario rerio（斑马鱼）中存在的两个 mcoln1 基因。我们的模型成功再现了 MLIV 患者中观察到的视网膜和神经肌肉缺陷，表明该模型适合研究疾病发病机制。重要的是，我们的模型揭示了对 MLIV 病理学起源和进展的新见解，包括自噬体积累对肌肉营养不良的贡献以及 mcoln1 在胚胎发育、毛细胞活力和细胞维持中的作用。鉴于斑马鱼适合大规模体内药物筛选，因此在斑马鱼中生成 MLIV 模型尤其重要，从而为治疗发现提供了新的机会。 庞贝病，一种严重的肌肉萎缩性疾病，其特征是溶酶体功能改变。严重的肌肉萎缩是庞贝病的一个标志，庞贝病是一种罕见的遗传性疾病，由酸性 α 葡萄糖苷酶 (GAA) 缺乏引起，GAA 是一种在溶酶体内将糖原分解为葡萄糖的酶。缺乏这种酶会导致婴儿迅速致命的心肌病和骨骼肌肌病；低水平的残余酶活性与儿童和成人发病的进行性骨骼肌肌病（通常不涉及心脏）有关。最近，我们发现患病肌肉细胞中 mTOR 信号传导失调，并专注于识别治疗干预的潜在位点。重要的是，通过 TSC 敲低或补充精氨酸重新激活 Pompe 小鼠整个肌肉中的 mTOR，导致萎缩逆转并显着消除自噬积聚。 庞贝氏症唯一可用的疗法是使用人重组 GAA 的酶替代疗法 (ERT)。这种疗法可以恢复心脏功能，但其对骨骼肌的影响要弱得多。庞贝氏骨骼肌中大量自噬的积累会对重组酶的运输和溶酶体递送产生负面影响。由于我们表明庞贝氏骨骼肌中 mTORC1 活性的恢复可显着减少自噬体积累，因此我们评估了当自噬积累被去除或减少时 ERT 是否可以更有效地发挥作用。为此，我们使用了双重方法，其中通过 TSC 消耗恢复 mTORC1 活性与 ERT 相结合。值得注意的是，这种方法导致肌肉质量增加和糖原积累减少，表明溶酶体病理学的逆转。因此，我们提出 TSC 介导的 mTOR 激活与 ERT 的组合可能有可能解决疾病病理学的多个方面。 改善庞贝氏症患者治疗的另一种方法是设计更有效的重组 GAA。我们与 Amicus Therapeutics 合作，测试了一种新型 rhGAA (ATB200)，它具有更高的 M6P 含量，从而改善与 CIM6PR 的相互作用以及向靶组织的递送。在小鼠庞贝氏症模型中注射 ATB200 与防止活性丧失和变性的小分子药理学伴侣一起，显着逆转了溶酶体内糖原的积累和自噬的积累，从而改善了肌肉功能。