Lysosomal Diseases

IV型黏液脂蛋白沉积症(MLIV)是一种常染色体隐性遗传病，以急性精神运动迟缓、精神萎缩症和视觉异常为特征，包括视网膜变性、角膜混浊、视神经萎缩和斜视。MLIV患者的大部分组织中均可见溶酶体包涵体。储存材料的组成是异质的，包括形成特征的多同心片层的脂类和粘多糖，以及可溶的颗粒状蛋白质。MLIV是由粘蛋白-1(MCOLN1，也称为TRPML1)突变引起的，MCOLN1是一种内溶酶体阳离子通道，属于瞬时受体电位(Trp)超家族离子通道。全细胞膜片钳和天然内溶酶体膜的记录表明，MCOLN1是一种内向(从管腔到细胞质)的整流通道，可以渗透到钙、钠、钾和Fe2/Mn2，其活性在低pH值下被增强。为了更好地了解这种疾病的病理，我们使用基因组编辑敲除了斑马鱼Dario rerio(斑马鱼)中存在的两个mcoln1基因。我们的模型成功地复制了MLIV患者中观察到的视网膜和神经肌肉缺陷，表明该模型适合于研究疾病的发病机制。重要的是，我们的模型揭示了MLIV病理的起源和发展的新见解，包括自噬小体积累对肌肉营养不良的贡献，以及mcoln1在胚胎发育、毛细胞活性和细胞维持中的作用。考虑到斑马鱼适合进行大规模体内药物筛选，在斑马鱼中产生MLIV模型特别重要，从而为治疗发现提供了新的机会。 庞培病，一种严重的肌肉萎缩障碍，其特征是溶酶体功能改变。严重的肌肉萎缩是庞培病的一个特征，这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。酸性α-葡萄糖苷酶是一种在溶酶体中将糖原分解为葡萄糖的酶。缺乏这种酶会导致婴儿迅速发生致命的心肌病和骨骼肌病；低水平的残留酶活性与儿童和成人发病的进行性骨骼肌病有关，通常没有心脏受累。最近，我们在患病的肌肉细胞中发现mTOR信号的异常调节，并将重点放在识别潜在的治疗干预部位上。重要的是，通过TSC基因敲除或补充精氨酸，在庞培小鼠的整个肌肉中重新激活mTOR，导致萎缩的逆转和自噬积聚的显著消除。 Pompe病的唯一有效治疗方法是使用人重组GAA的酶替代疗法(ERT)。这种疗法可以恢复心脏功能，但对骨骼肌的效果要差得多。庞贝骨骼肌中大量的自噬堆积对重组酶的运输和溶酶体递送产生了负面影响。由于我们证明了庞贝骨骼肌mTORC1活性的恢复显著地减少了自噬小体的积累，因此我们评估了当自噬积聚被移除或减少时，ERT是否可能更有效地工作。为此，我们使用了双重方法，其中通过TSC耗竭恢复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聚减少，提示溶酶体病理逆转。因此，我们提出，TSC介导的mTOR激活与ERT的结合可能具有解决疾病病理的多个方面的潜力。 改善Pompe患者治疗的另一种方法是设计一种更有效的重组GAA。在与Amicus Treeutics的合作下，我们测试了一种新型的rhGAA(ATB200)，它具有显著更高的M6P含量，从而改善了与CIM6PR的相互作用并将其输送到靶组织。在小鼠Pompe模型中注射ATB200，结合防止活性丧失和变性的小分子药理伴侣，显著逆转溶酶体内糖原积聚和自噬积聚，导致肌肉功能改善。

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.