Abstract

Lysosomal storage diseases (LSDs) encompass around 50 different inherited syndromes. They are caused by deficiencies in lysosomal enzymes or transporters, resulting in intra-lysosomal buildup of undegraded metabolites (1). Gangliosidosis correspond to a sub-set of LSDs characterized by the accumulation of gangliosides, which are complex glycolipids containing ceramide linked to a variable number of saccharides and sialic acid residues (2). Hydrolysis of GM2 occurs in the lysosome, and it requires the proper function of HEXA and HEXB. Inheritable mutations impairing either gene result in the intracellular buildup of GM2, causing the devastating disorders known as Tay-Sachs and Sandhoff diseases respectively (3) Cellular clearance is controlled by the Transcription Factor EB (TFEB), which is the master regulator of the “Coordinated Lysosomal Expression and Regulation (CLEAR)” network. Under normal conditions TFEB resides in the cytoplasm, while under stress conditions, TFEB translocates to the nucleus, where it activates the transcription of genes encoding proteins involved in a wide range of functions related to lysosomal cellular clearance, such as lysosomal biogenesis, autophagy, exocytosis and phagocytosis (4). Up to date, there are no proven disease-modifying therapies for the gangliosidoses (2). Substrate reduction therapies, in addition to gene therapies and neural stem cells transplants have been successfully explored in animal models as potential strategies for treating GM2 Gangliosidosis (5, 6). At the cellular level, it has been shown that sucrose, an activator of TFEB translocation, enhance the activity of a mutated HexA in cells, presumably by augmenting the pool of mutated enzyme and its processing through the secretory pathway (7). However, the direct role of TFEB and other pathways in promoting clearance in GM2 gangliosidosis has not been explored. Study Goals The goal of this grant proposal is to promote cellular clearance in in vitro models of GM2 gangiosidosis. This will be achieved by taking advantage of a chemical inhibitor of HEXA/B and the unbiased methods to study and quantify lysosomal related cellular phenotypes developed by Diego Medina, co-PI in this application, at the High Content Screening (HSC) facility at TIGEM. This grant will provide unique information about the role of Tfeb and other genes in cellular clearance, and potential novel targets for treating GM2 accumulation.