What else is in store for autophagy? Exocytosis of autolysosomes as a mechanism of TFEB-mediated cellular clearance in Pompe disease

Abstract

It is hard to find an area of biology in which autophagy is not involved. In fact, the topic extends beyond scientific research to stimulate intellectual exercise and entertainment—autophagy has found its way into a crossword puzzle (Klionsky, 2013). We have found yet another function of autophagy while searching for a better treatment for Pompe disease, a devastating metabolic myopathy resulting from excessive lysosomal glycogen storage. To relieve this glycogen burden, we stimulated lysosomal exocytosis through upregulation of transcription factor EB (TFEB). Overexpression of TFEB in Pompe muscle clears the cells of enlarged lysosomes, reduces glycogen levels, and alleviates autophagic buildup, the major secondary abnormality in Pompe disease. Unexpectedly, the process of exocytosis does not seem to be a purely lysosomal event; vesicles arranged along the plasma membrane are double-labeled with the lysosomal marker LAMP1 and the autophagosomal marker LC3, indicating that TFEB induces the exocytosis of autolysosomes. Furthermore, the effects of TFEB are almost abrogated in autophagy-deficient Pompe mice, suggesting a previously unrecognized role of autophagy in TFEB-mediated cellular clearance.

Pompe disease, a fatal cardiac and skeletal muscle myopathy, is caused by a deficiency of acid α-glucosidase (GAA), the enzyme responsible for lysosomal degradation of glycogen. The disease strikes at different ages and ranges from severe infantile to relatively attenuated adult forms. The currently available therapy, designed to provide the missing enzyme (enzyme replacement therapy or ERT), proves to be very successful in reversing cardiac but not skeletal muscle abnormalities. This failure is at least partially attributed to dysfunctional autophagy, which greatly contributes to the pathological cascade in skeletal muscle. Indeed, we have previously shown that muscle-specific genetic suppression of autophagy allows for successful reversal of lysosomal pathology by ERT. These proof-of-principle experiments in autophagy-deficient Pompe mice clearly indicated a need for autophagy-targeted therapy.

In searching for such a therapy, we initially addressed the mechanism behind the massive accumulation of autophagosomes in the core of Pompe muscle fibers. For these experiments, we used transgenic Pompe mice in which autophagosomes are labeled with GFP-LC3 (GFP-LC3:Pompe). Time-lapse confocal imaging of live fibers transfected with mCherry-LAMP1 (lysosomal marker) revealed little if any fusion between autophagosomes and lysosomes in the buildup area. This impaired fusion, a condition known as autophagic block, appears to be responsible for the autophagic buildup. The question was how to restore vesicular fusion.

Our previous data demonstrated that lysosomal-autophagosomal fusion can be stimulated by transcription factor EB (TFEB); furthermore, TFEB promotes lysosomal exocytosis leading to cellular clearance in several models of lysosomal storage disorders, including multiple sulfatase deficiency and mucopolysaccharidosis type IIIA. These two aspects of TFEB function seemed ideally suited to both resolve autophagic buildup and rid muscle cells of distended glycogen-filled lysosomes—the hallmarks of Pompe disease.

First, we addressed lysosomal pathology to see if TFEB would have similar success in terminally differentiated muscle cells, the tissue most resistant to ERT. The number of enlarged lysosomes was significantly reduced in Pompe muscle fibers transfected with GFP-TFEB. In addition, glycogen levels dramatically decreased and approached wild-type levels in whole muscle infected with adeno-associated virus (AAV)-TFEB. Next, to evaluate fusion events within areas of autophagic accumulation, we once again turned to time-lapse confocal imaging of live fibers from GFP-LC3:Pompe mice. Muscle was transfected with mCherry-LAMP1 and Flag-TFEB (in this experiment, mCherry served as a reporter for both). Strikingly, we did not see any of the massive autophagic buildup that is so prominent in untreated GFP-LC3 fibers. The missing buildup was a mystery, but the emergence of many GFP-LC3 puncta after hours of confocal exposure—surges in autophagy—provided one clue. Using these surges as a surrogate for the study of vesicular fusion, we found that lysosomes and autophagosomes readily merged in TFEB-treated fibers, resulting in near complete colocalization of red and green after hours of microscopy. In fact, vesicles aligned at the plasma membrane—as if ready for exocytosis—were not pure lysosomes, but were instead autolysosomes, a product of fusion between LAMP1- and LC3-postive vesicles.

In thinking about this autolysosome alignment, we began to wonder if autophagosomal-lysosomal fusion could be a prerequisite for TFEB-mediated exocytosis in muscle tissue. Lysosomal function is known to depend on the ability of lysosomal membranes to fuse with other vesicles’ membranes and with the plasma membrane. Perhaps the merging of lysosomal and autophagosomal membranes provides machinery to facilitate fusion with the plasma membrane, the key step in exocytosis.

If this speculation is true and lysosomes rely on autophagosomes to undergo exocytosis, then the process of TFEB-mediated clearance would be blunted in the setting of genetically suppressed autophagy. Here, we put our autophagy-deficient Pompe mice to good use and followed the movement of mCherry-LAMP1-labeled lysosomes in fibers expressing GFP-TFEB. We did see characteristic signs of TFEB’s effects—redistribution and alignment of LAMP-positive vesicles along the plasma membrane—but TFEB-induced clearance of enlarged lysosomes was significantly diminished.

The autophagosome has generally been looked upon as a vesicle fully dependent on the lysosome for fulfillment of its function—delivery of macromolecules and worn-out organelles for degradation. But perhaps this relationship is not so one-sided. Our data suggest that at least in some settings the lysosome depends on the autophagosome for efficient TFEB-mediated exocytosis. When our paper was in press, Zhou et al. (Cell Research, 2013) claimed that under conditions of starvation or MTORC1 inhibition, the activation of lysosomal function (i.e., acidification, cathepsin activity, and proteolysis) also requires fusion with autophagosomes. It remains to be seen if indeed autophagosomes are so critical for the proper functioning of the lysosome, but what is clear is that the relationship between these two vesicular structures is more complex than originally appreciated.

Spampanato C, Feeney E, Li L, Cardone M, Lim JA, Annunziata F, et al. Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. EMBO Mol Med. 2013;5:691–706. doi: 10.1002/emmm.201202176.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.