Lysosomal Diseases

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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 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. More recently, we have looked at the long-term effect of AT-GAA on a range of lysosomal/autophagic markers, mTORC1-dependent and independent protein synthesis and proteasomal degradation, as well as on the upstream regulators and downstream targets of AMPK and mTORC1. Our data indicate that the therapeutic benefit of AT-GAA is clear; the therapy significantly improved or reversed multiple aspects of the disease pathogenesis, thus offering clear advantage over the available therapy.