Substrate localisation as a therapeutic option for Pompe disease.

Abstract

Pompe disease is a progressive form of muscular dystrophy caused by a deficiency in the lysosomal enzyme α-glucosidase (GAA). GAA catabolises glycogen and its deficiency leads to glycogen accumulation in the vesicular network of affected cells. Multiple therapies exist to treat Pompe disease but these are not completely effective (Winkel et al., 2003), necessitating the development of new therapeutic strategies. A number of enzymes that reside outside of the lysosome, either in the cytoplasm (Watanabe et al., 2008) or in circulation (Ugorski et al., 1983), can catabolise glycogen. It was postulated that if vesicular glycogen in Pompe cells was transferred out of these compartments it could then be alternatively degraded. The ability to remove vesicular glycogen from Pompe cells may reduce the onset/progression of the disorder, providing a therapeutic option for patients. Exocytosis is a ubiquitous cellular mechanism where intracellular vesicles fuse with the cell surface and permit vesicle content to be released from the cell. It was postulated that exocytosis may provide a mechanism to release accumulated glycogen from Pompe cells. Approximately 4% of vesicular glycogen was exocytosed from Pompe skin fibroblasts after 2 hrs in culture. Pompe cells exocytosed 2.7-fold more glycogen than unaffected cells. A cellular mechanism was therefore identified that had the capacity to release glycogen from Pompe cells. Culture conditions can alter the amount of exocytosis in fibroblasts (Martinez et al., 2000). In this study the effect of cell confluence and components of the culture media on lysosomal exocytosis was examined in Pompe skin fibroblasts. Increasing the extracellular concentration of Ca²⁺ led to a 1.4-fold increase in glycogen release compared to cells cultured in standard media conditions. Culture confluence had a key influence on glycogen exocytosis, with sub-confluent Pompe cells releasing >80% of glycogen after 2 hrs in culture, 35-fold higher than confluent cells. Exocytic mechanisms therefore exist that allow up-regulation of glycogen exocytosis in Pompe skin fibroblasts. A number of pharmacological compounds induce exocytosis in cultured cells (Amatore et al., 2006). Pompe skin fibroblasts treated with three compounds; calcimycin, lysophosphatidylcholine and α-L-iduronidase, each demonstrated a ≥ 1.5-fold increase in glycogen exocytosis, when compared to untreated Pompe controls. Calcimycin was the most effective compound for inducing glycogen exocytosis, with 12% released after 2 hrs of treatment, but confluent Pompe cells released less than that observed from sub-confluent Pompe cells. This difference in glycogen release may have resulted from the induction of different exocytic mechanisms. Complete exocytosis, where the vesicle completely fuses with the cell surface and releases all vesicle content, is induced in sub-confluent Pompe cells. In contrast, cavicapture, involving only a partial pore opening and limited vesicle content release, is induced in response to calcimycin treatment. The identification of a compound capable of inducing complete exocytosis may therefore improve glycogen release from Pompe cells. Taken together, natural glycogen exocytosis and the ability to induce glycogen exocytosis with pharmacological compounds provided proof-of-concept for exocytic induction as a strategy to re-locate accumulated glycogen from Pompe cells, potentially providing a new therapeutic option for the disorder.