Monica Cardone

A recently proposed therapeutic approach for lysosomal storage disorders (LSDs) relies upon the ability of transcription factor EB (TFEB) to stimulate autophagy and induce lysosomal exocytosis leading to cellular clearance. This approach is particularly attractive in glycogen storage disease type II [a severe metabolic myopathy, Pompe disease (PD)] as the currently available therapy, replacement of the missing enzyme acid alpha-glucosidase, fails to reverse skeletal muscle pathology. PD, a paradigm for LSDs, is characterized by both lysosomal abnormality and dysfunctional autophagy. Here, we show that TFEB is a viable therapeutic target in PD: overexpression of TFEB in a new muscle cell culture system and in mouse models of the disease reduced glycogen load and lysosomal size, improved autophagosome processing, and alleviated excessive accumulation of autophagic vacuoles. Unexpectedly, the exocytosed vesicles were labelled with lysosomal and autophagosomal membrane markers, suggesting that TFEB induces exocytosis of autophagolysosomes. Furthermore, the effects of TFEB were almost abrogated in the setting of genetically suppressed autophagy, supporting the role of autophagy in TFEB-mediated cellular clearance.

The mechanistic target of rapamycin (mTOR) serine/threonine kinase, a critical regulator of cell proliferation, is frequently deregulated in human cancer. Although rapamycin inhibits the two canonical mTOR complexes, mTORC1 and mTORC2, it often shows minimal benefit as an anticancer drug. This is caused by rapamycin resistance of many different tumors, and we show that a third mTOR complex, mTORC3, contributes to this resistance. The ETS (E26 transformation-specific) transcription factor ETV7 interacts with mTOR in the cytoplasm and assembles mTORC3, which is independent of ETV7's transcriptional activity. This complex exhibits bimodal mTORC1/2 activity but is devoid of crucial mTORC1/2 components. Many human cancers activate mTORC3 at considerable frequency, and tumor cell lines that lose mTORC3 expression become rapamycin-sensitive. We show mTORC3's tumorigenicity in a rhabdomyosarcoma mouse model in which transgenic ETV7 expression accelerates tumor onset and promotes tumor penetrance. Discovery of mTORC3 represents an mTOR paradigm shift and identifies a novel target for anticancer drug development.

Mucopolysaccharidosis type II (MPSII; Hunter syndrome) is a lysosomal storage disorder caused by a deficiency in the enzyme iduronate 2-sulfatase (IDS). At present, the therapeutic approaches for MPSII are enzyme replacement therapy and bone marrow transplantation, although these therapies have some limitations. The availability of new AAV serotypes that display tissue-specific tropism and promote sustained expression of transgenes offers the possibility of AAV-mediated gene therapy for the systemic treatment of lysosomal diseases, including MPSII. We have characterized in detail the phenotype of IDS-deficient mice, a model of human MPSII. These mice display a progressive accumulation of glycosaminoglycans (GAGs) in many organs and excessive excretion of these compounds in their urine. Furthermore, they develop skeleton deformities, particularly of the craniofacial bones, and alopecia, they perform poorly in open-field tests and they have a severely compromised walking pattern. In addition, they present neuropathological defects. We have designed an efficient gene therapy approach for the treatment of these MPSII mice. AAV2/8TBG-IDS viral particles were administrated intravenously to adult MPSII mice. The plasma and tissue IDS activities were completely restored in all of the treated mice. This rescue of the enzymatic activity resulted in the full clearance of the accumulated GAGs in all of the tissues analyzed, the normalization of the GAG levels in the urine and the correction of the skeleton malformations. Overall, our findings suggest that this in vivo gene transfer approach has potential for the systemic treatment of patients with Hunter syndrome.

BACKGROUND: Pompe disease (PD) is a metabolic myopathy caused by alpha-glucosidase (GAA) deficiency and characterized by generalized glycogen storage. Heterogeneous GAA gene mutations result in wide phenotypic variability, ranging from the severe classic infantile presentation to the milder intermediate and late-onset forms. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA), the only treatment available for PD, intriguingly shows variable efficacy in different PD patients. To investigate the mechanisms underlying the variable response to ERT, we studied cell morphology of PD fibroblasts, the distribution and trafficking of the cation-independent mannose-6-phosphate receptor (CI-MPR) that mediates rhGAA uptake, and rhGAA uptake itself. RESULTS: We observed abnormalities of cell morphology in PD cells. Electron microscopy analysis showed accumulation of multivesicular bodies and expansion of the Golgi apparatus, and immunolocalization and western blot analysis of LC3 showed activation of autophagy. Immunofluorescence analysis showed abnormal intracellular distribution of CI-MPR in PD fibroblasts, increased co-localization with LC3 and reduced availability of the receptor at the plasma membrane. The recycling of CI-MPR from the plasma membrane to the trans-Golgi network was also impaired. All these abnormalities were more prominent in severe and intermediate PD fibroblasts, correlating with disease severity. In severe and intermediate PD cells rhGAA uptake and processing were less efficient and correction of GAA activity was reduced. CONCLUSION: These results indicate a role for disrupted CI-MPR trafficking in the variable response to ERT in PD and have implications for ERT efficacy and optimization of treatment protocols.