The major limitation of the current therapy is the difficulty of the enzyme to reach skeletal muscle and its preferential uptake by the liver. We have shown that on top of that, the trafficking and delivery of the replacement enzyme to the lysosomes in skeletal muscle are negatively affected by the presence of massive autophagic buildup and large lipofuscin deposits within the areas of autophagic accumulation. A critical step in the autophagic pathway, which is profoundly impaired in Pompe muscle, is the fusion between autophagosomes and lysosomes where the contents of autophagosomes is digested and recycled. Importantly, both pathologies, autophagic buildup and lipofuscin inclusions, are not amenable to ERT. An entirely novel approach has recently been proposed for treatment of lysosomal storage disorders, which relies on the ability of the DNA-binding transcription factors EB (TFEB) and E3 (TFE3) to stimulate autophagosomal-lysosomal fusion and induce lysosomal exocytosis (expulsion of the lysosomal content outside the cell) leading to cellular clearance. We have demonstrated that overexpression of TFEB or TFE3 in Pompe muscle cells reduced the size of LAMP-positive lysosomes, decreased the amount of accumulated glycogen, and alleviated autophagic buildup. These data established TFEB/TFE3 as valid therapeutic targets in Pompe disease. This approach circumvents the inefficient enzyme delivery to skeletal muscle and restores autophagic flux. In addition, we have identified a compound that induces nuclear translocation (activation) of endogenous TFE3 and demonstrated that two kinases, mTORC1and MAPK (ERK1/2), are involved in the regulation of TFEB and TFE3 in skeletal muscle. Both defective autophagy and accelerated production of lipofuscin pointed to the mitochondrial abnormalities in Pompe skeletal muscle. The autophagic pathway is responsible for the removal of worn-out and damaged mitochondria, a process known as mitophagy. Indeed, we have found multiple mitochondrial defects in mouse and human models of Pompe disease. ChIP-seq analysis of C2C12 cells with specific anti-TFE3 antibody showed the similarity to the previously established TFEB-binding sites in other cells. In addition, TFE3 binding in muscle cells was strongly associated with mitochondrial genes; inspection of several mitochondria-related genes revealed the presence of E-box sequences in their promoters. These data suggested that upregulation of TFE3 may have an additional benefit by stimulating mitochondrial biogenesis in Pompe muscle cells. To further explore the connection between TFE3 and mitochondrial genes and to gain insight into the functional significance of the TFE3 binding in muscle cells, we performed mRNA-seq of non-transfected, Ad-TFE3-transfected, and TFE3 siRNA-transfected C2C12 myoblasts. These data were overlapped with the Chip-seq data (comparing the sets of genes with TFE3 peaks within 1 kb from the TSS); the overlap was represented by 169 genes associated with TFE3 overexpression, and by 211 genes associated with TFE3 silencing. Using MitoCarta 2.0, as a reference, we have identified a reliable list of TFE3-induced mitochondria-related genes in myoblasts, and selected genes were verified by western analysis. Thus, we have identified mitochondrial genes as new TFE3-target genes in myoblasts. Overexpression of Ad-TFE3 in C2C12 cells and in immortalized KO myotubes significantly enhanced mitochondrial mass as evidenced by the increased level of the mitochondrial marker, COXIV. Our second major project included analysis of the mTORC1 signaling pathway in Pompe skeletal muscle. The evaluation of the mTORC1 status is particularly relevant to Pompe disease, because it is a muscle wasting disorder, and mTORC1 is directly involved in the control of muscle mass. The signaling pathways responsible for the loss of muscle mass in Pompe disease are largely unknown, and the limited data in the literature on the subject are conflicting. Understanding the mechanism of the disturbed mTOR signaling in Pompe muscle cells opens the possibility for much needed novel treatment strategies. We anticipated that lysosomal enlargement and the acidification defect (as we have shown previously in Pompe muscle cells) would affect the interaction of the components of a complex machinery involved in the recruitment of mTORC1 to the lysosome (activation) and its release from the lysosome (inactivation). Therefore, we systematically analyzed mTOR pathway by looking at the mTOR downstream targets and the upstream inputs in Pompe muscle cells. We conducted an extended analysis of mTOR pathway in Pompe muscle cells by evaluating mTOR activity, localization, regulation in response to nutrients, and its role in the control of protein synthesis and autophagy. This study is the first systematic analysis of mTORC1 signaling in Pompe muscle cells. Based on the extensive experimental data, we proposed a model of mTOR dysregulation in Pompe disease. Most importantly, we have identified sites of therapeutic intervention and used targeted approaches to reinstate mTOR activity in Pompe muscle cells: 1) manipulation of v-ATPase activity by addressing the lysosomal acidification defect to force proper mTORC1 localization; and 2) manipulation of TSC2 to relieve its inhibitory effect on mTOR. Recent data demonstrated that arginine can activate mTORC1 by suppressing lysosomal localization of the TSC2 complex. Considering the site of arginine action, this amino acid seems ideally suited for correction of the defect in KO cells. Most exciting, we could reinstate mTOR activity in KO cells and in KO mice by providing excess of L-arginine. The attractiveness of this approach is obvious in that this amino acid can be taken as a natural dietary supplement. This safe and effective treatment strategy may have broad relevance for a large group of metabolic, neuromuscular, and lysosomal storage disorders. In addition, we have established a collaboration with Amicus Therapeutics, a biotechnology company, which is developing novel enzyme replacement therapy for Pompe disease. We have tested the new replacement enzyme in our mouse model of the disease, and we have found that the new compound works far better than the currently available drug.
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