Lysosomal Diseases

IV型粘脂沉积症（MLIV）是一种常染色体隐性遗传疾病，其特征为急性精神发育迟滞、瞳孔缺乏和视觉异常，包括视网膜变性、角膜混浊、视神经萎缩和斜视。在MLIV患者的大多数组织中发现溶酶体包涵体。储存材料的组成是异质的，包括形成特征性多同心薄片的脂质和粘多糖，以及可溶性颗粒蛋白质。MLIV是由粘磷脂-1（MCOLN 1，也称为TRPML 1）突变引起的，MCOLN 1是一种属于离子通道瞬时受体电位（TRP）超家族的内溶酶体阳离子通道。全细胞膜片钳，以及记录的天然内溶酶体膜，表明MCOLN 1的功能作为一个向内（从内腔到细胞质）整流通道渗透Ca 2+，Na+，K+和Fe 2 +/Mn 2+，其活性是由低pH值增强。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两个mcoln 1基因存在于达里奥鱼（斑马鱼）。我们的模型成功地再现了MLIV患者中观察到的视网膜和神经肌肉缺陷，表明该模型适用于研究疾病发病机制。重要的是，我们的模型揭示了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mcoln 1在胚胎发育，毛细胞活力和细胞维持中的作用。在斑马鱼中产生MLIV模型是特别相关的，因为这种生物体适合于大规模体内药物筛选，从而为治疗发现提供了新的机会。 庞贝氏症，一种以溶酶体功能改变为特征的严重肌肉萎缩性疾病。严重的肌肉萎缩是庞贝氏症的一个标志，庞贝氏症是一种罕见的遗传性疾病，由缺乏酸性β-葡糖苷酶（GAA）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;低水平的残留酶活性与儿童和成人发作的进行性骨骼肌肌病相关，通常不涉及心脏。最近，我们在患病的肌肉细胞中发现了mTOR信号转导的失调，并专注于识别治疗干预的潜在位点。重要的是，通过TSC敲低或精氨酸补充，庞贝氏症小鼠的整个肌肉中mTOR的再活化导致萎缩的逆转和自噬积累的显著去除。 庞贝氏症的唯一可用疗法是使用人重组GAA的酶替代疗法（ERT）。这种疗法可以恢复心脏功能，但其在骨骼肌中的作用要弱得多。庞贝氏症骨骼肌中的大量自噬积聚对重组酶的运输和溶酶体递送产生负面影响。由于我们发现庞贝氏症骨骼肌中mTORC 1活性的恢复显著降低了自噬体的积累，我们评估了当自噬积累被去除或减少时，ERT是否可以更有效地工作。为此，我们使用了双重方法，其中通过TSC耗竭恢复mTORC 1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，表明溶酶体病理学逆转。因此，我们提出TSC介导的mTOR激活与ERT的组合可能具有解决疾病病理学的多个方面的潜力。 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。与Amicus Therapeutics合作，我们测试了一种新型rhGAA（ATB 200），其M6 P含量显著更高，从而改善了与CIM 6PR的相互作用并递送至靶组织。在小鼠庞贝氏症模型中注射ATB 200以及可防止活性丧失和变性的小分子药理学伴侣，可显著逆转溶酶体内糖原蓄积和自噬积聚，从而改善肌肉功能。

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.