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. Numerous lysosomes and autolysosomes loaded with lipofuscin - an indigestible autofluorescent lipopigment - in skeletal muscle appear to be a hallmark of the disease in children and adults. In some patients more than 75% of fibers contained these structures, which can span up to several hundred microns along the length of the fiber. Lipofuscin accumulation - a result of inefficient lysosomal degradation - may in turn exacerbate both lysosomal and autophagic abnormalities. 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 factor EB (TFEB) to induce lysosomal exocytosis (expulsion of the lysosomal content outside the cell) leading to cellular clearance. This approach is particularly attractive for Pompe disease since TFEB was also shown to stimulate autophagosomal-lysosomal fusion. Indeed, we have demonstrated that TFEB-overexpression in Pompe muscle cells (both in mice and in culture) reduced the size of LAMP-positive lysosomes, decreased the amount of accumulated glycogen, and alleviated autophagic buildup. These data established TFEB as a valid therapeutic target in Pompe disease. More recent studies performed in our laboratory showed that a closely related but distinct transcription factor E3 (TFE3) is a more attractive target since it is abundant in skeletal muscle, whereas TFEB is barely detectable. Experiments in Pompe muscle cells demonstrated that similar to TFEB, overexpression of TFE3 induced lysosomal exocytosis and promoted glycogen clearance. By using CHIP-sequencing method we have shown that TFE3 binds to the promoter region of many lysosomal and autophagic genes in muscle cells (C2C12). These experiments are done in collaboration with Dr. Sartorelli. Furthermore, we have discovered a novel role for TFE3 in the regulation of mitochondrial biogenesis in muscle cells. In addition, we have identified two compounds which induce nuclear translocation (activation) of endogenous TFE3. Similar to TFEB, TFE3 activity is regulated by phosphorylation. In its phosphorylated form TFE3 is inactive and localized in the cytosol; inhibition of phosphorylation (for example, by nutrient deprivation) activates TFE3 and promotes its translocation to the nucleus where it stimulates the expression of multiple target genes. Therefore, pharmacological inhibition of TFE3 phosphorylation would promote cellular clearance in Pompe disease as well as in other lysosomal storage disorders. Two kinases, mTORC1 and ERK, have been implicated in TFEB regulation in different cells; however, the regulation of TFE3 in skeletal muscle remains an open question. By using mTORC1 and ERK inhibitors in Pompe cell cultures we have demonstrated that both mTORC1and MAPK (ERK1/2) kinases are involved in the regulation of TFEB and TFE3 in this tissue. To evaluate the regulatory role of TFE3 in Pompe disease we have used ChIP-seq to generate genome-wide maps of TFE3 in the diseased and control muscle cells (unstimulated and starved). The inspection of mapped sequence reads (using a genome browser) from the comparative ChIP-seq analysis revealed a striking difference in the number, strength, and location of peaks (regions of high sequencing read density) between Pompe and control cells, thus providing a snapshot of the disease-specific pattern of TFE3-mediated gene regulation. We are currently analyzing these data. The appeal of TFEB or TFE3 modulation as a therapeutic option in Pompe disease is twofold: 1) this approach circumvents the major hurdle of the current therapy, namely inefficient enzyme delivery to skeletal muscle; and 2) unlike other efforts, it restores autophagic flux and addresses both lysosomal and autophagic pathologies. 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. Mitochondria-induced oxidative stress and flawed autophagy are common features of neurodegenerative and lysosomal storage diseases (LSDs). Although defective autophagy is particularly prominent in Pompe disease, mitochondrial function has escaped examination in this typical LSD. We have used several models to examine mitochondrial defects in Pompe disease: GAA-KO mice; previously developed immortalized GAA-deficient muscle cells; a newly generated GAA-KO cell line, in which lysosomes are labeled with mCherry-LAMP1; and human primary muscle cells derived from Pompe patients with adult form of the disease. We have found multiple mitochondrial defects in mouse and human models of Pompe disease, including a profound dysregulation of Ca2+ homeostasis, mitochondrial Ca2+ overload, an increase in reactive oxygen species, a decrease in mitochondrial membrane potential, and an increase in caspase-independent apoptosis, as well as a decreased oxygen consumption and ATP production of mitochondria. In addition, gene expression studies revealed a striking upregulation of beta 1 subunit of L-type Ca2+ channel in Pompe muscle cells. We have demonstrated that disturbance of Ca2+ homeostasis and mitochondrial abnormalities in Pompe disease represent early changes in a complex pathogenic cascade leading from a deficiency of a single lysosomal enzyme to severe and hard-to-treat autophagic myopathy. Most importantly, we have shown that two Ca2+ channel blockers verapamil and nifedipine - both approved hypertension drugs - reversed the mitochondrial abnormalities in the KO cells, indicating that a similar approach can be beneficial to the plethora of lysosomal and neurodegenerative disorders. In addition, we have analyzed mTORC1 signaling pathway in Pompe skeletal muscle. mTORC1 signaling is particularly important in Pompe disease because it is a muscle wasting disorder and mTORC1 is a major regulator of cell metabolism directly involved in the control of muscle mass.
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