TFEB and the CLEAR network

Abstract

The general view of the lysosome as the terminal end of cellular catabolic pathways, has started to change due to the recent discoveries of a lysosomal nutrient sensing machinery and of a lysosome-to-nucleus signaling mechanism that modulate lysosomal function by way of the master transcriptional regulator TFEB. Lysosomal biogenesis and autophagy are coordinated by TFEB, whose function is regulated by phosphorylation. TFEB interacts with and is phosphorylated by mTORC1 at the lysosomal surface. Thus, conditions resulting in inhibition of mTOR, such as starvation and lysosomal stress, promote TFEB nuclear translocation. Preliminary evidences showing that the TFEB activation are able to ameliorate the phenotype of lysosomal storage disorders and more common neurodegenerative diseases have opened an extraordinary possibility for the development of innovative therapies. Research in TFEB and lysosomal function has continued to advance and attract interest due to increased understanding of the mechanisms behind lysosomal function. In this paper, we present a set of procedures that facilitate examination of TFEB function and its related processes.

Introduction

In order to function properly and adapt under different physiological and pathological conditions, the lysosomal compartment requires the coordinated expression and action of various components, such as acid hydrolases, lysosomal acidification machinery, and membrane proteins. Thorough examination of publicly available microarray data, followed by coexpression analysis of lysosomal genes, both indicated that lysosomal genes have a statistically significant tendency to be coexpressed in various different tissues and cell types, under varying conditions. Consistently, pattern discovery analysis revealed that a palindromic 10-base site can be found in the promoters of several lysosomal genes. This 10-base sequence was recognized as an E-box, which is the target site for basic helix-loop-helix transcription factors. These two independent approaches led to the discovery of the lysosomal gene network—the coordinated lysosomal expression and regulation (CLEAR) network—and of its master regulator TFEB, a member of the microphthalmia-associated transcription factor (MITF) subfamily of transcription factors. TFEB positively regulates the expression of lysosomal genes, controls lysosome population and promotes cellular degradation of lysosomal substrates (Palmieri et al., 2011, Sardiello et al., 2009). Additionally, TFEB regulates autophagy, and consequently, its overexpression leads to a significant increase in autophagosome production in cultured cells. Elevated lysosome–autophagosome fusion and degradation of long-lived autophagy substrates were the direct results of increased TFEB activity (Settembre et al., 2011). Specifically, TFEB activates the transcription of genes that encode proteins involved in a range of operations related to cellular clearance, such as lysosomal biogenesis, autophagy, exocytosis, endocytosis, and additional lysosome-associated processes, such as lysosomal proteostasis, phagocytosis, immune response mechanisms, and lipid catabolism (Palmieri et al., 2011). Furthermore, in the context of clinical approaches to the treatment of lysosomal storage disorders (LSDs) and neurodegenerative conditions, the discovery of the CLEAR network may represent a paradigm shift, given that it presents the unprecedented opportunity of exploitation of the lysosomal system for the treatment of such disorders. Preliminary evidence has shown that TFEB overexpression reduces the accumulation of glycosaminoglycans (GAGs) and cellular vacuolization in glia-differentiated neuronal stem cells (NSCs) originating from mouse models of multiple sulfatase deficiency and mucopolysaccharidosis IIIA, two severe types of LSDs (Medina et al., 2011). Similar results were obtained in other types of LSDs, such as neuronal ceroid lipofuscinosis type 3 (Batten disease) and glycogen storage disease type II (Pompe disease) (Medina et al., 2011, Spampanato et al., 2013). Furthermore, similar approaches showed promising results in murine models of more common neurodegenerative diseases, such as Parkinson's disease, Huntington's disease, and Alzheimer's disease (Decressac et al., 2013, Polito et al., 2014, Tsunemi et al., 2012). Finally, a mutated form of α1-antitrypsin in a mouse model of liver fibrosis was cleared after overexpression of TFEB in hepatic tissue (Pastore et al., 2013).

The mechanism by which TFEB promotes the clearance of stored material needs to be further characterized. However, it is possible that TFEB-mediated cellular clearance results from a combination of the effects of lysosomal biogenesis, autophagy, and lysosomal exocytosis (Medina et al., 2011). Pharmacological modulation of lysosomal function via activation of TFEB is an attractive therapeutic strategy to promote cellular clearance in all of the aforementioned diseases. The results from recent work supports this alternative approach, demonstrating that a well-known candidate molecule for LSDs, Genistein, reduces GAG accumulation, in part, by inducing TFEB nuclear translocation (Moskot et al., 2014). Therefore, drug-screening approaches aimed at identifying molecules that promote TFEB nuclear translocation present an interesting path forward. The important implications resulting from the study of TFEB-mediated function prompted us to present a comprehensive set of the major experimental procedures used to examine TFEB signaling and lysosomal pathways (Figure 1).