Different experimental findings support a possible function of proteolytic processes in LD, and a role of the malin-laforin complex in the degradation of misfolded proteins by the ubiquitin-proteasome pathway has already been proposed. Therefore, we also considered the possibility of altered autophagy in LD. Autophagy sequesters a variety of proteins, lipids, carbohydrates and nucleic acids from the cytoplasm in autophagosomes, which are later degraded in lysosomes. Although glycogen is degraded in the cytosol by glycogen phosphorylase and the glycogen debranching enzyme, glycogen can be also degraded in the lysosomes via lysosomal acid alpha-glucosidase, also called acid maltase. The importance of this lysosomal pathway of glycogen degradation is illustrated by glycogen storage disease type II, also called Pompe disease, in which this single lysosomal enzyme is lacking.
Using laforin-deficient fibroblasts from LD patients and mouse embryonic fibroblasts (MEFs) and liver from epm2a-/- mice that replicate LD, we found decreased levels and impaired formation of LC3-II, indicating compromised autophagosome formation. We also found increased accumulation of polyubiquitinated proteins, which can result from a defect in both the ubiquitin-proteasome system and/or autophagy. This accumulation was even observed in the presence of proteasome inhibitors, suggesting that at least part of this accumulation is proteasome-independent. Finally, there was increased accumulation in liver extracts from laforin null mice of the autophagy substrate p62. However, laforin is not absolutely necessary for autophagy, since the lack of laforin only partially compromises long-lived autophagic protein clearance. Nevertheless, a partial deficit would be sufficient to contribute to many of the protein accumulation pathologies we have observed in these mouse models. In agreement with the laforin null data, overexpression of laforin increased the levels of LC3-II and reduced the amount of protein aggregates in an autophagy-dependent manner. Thus, it appears that laforin positively regulates autophagy.