Assistant Investigator, TIGEM
Head of High Content Screen Facility, TIGEM
Lysosomal calcium signalling and autophagy
Lysosomes are the end terminal for the degradation of cargo that arrives from different cellular routes such as the endocytic and the autophagic pathways. The extent of this catabolic activity depends on environmental cues such as nutrient availability. Thus, the lysosomes possess nutrient-sensing machinery consisting of mechanistic/mammalian target of rapamycin (mTOR), the master regulator of growth and a negative regulator of autophagy, and its associated proteins. Upon nutrient starvation mTOR signalling is inhibited while the master regulator of lysosomal function TFEB is activated. We have recently shown that lysosomal Ca2+ release through the non-selective cation channel TRPML1 plays a major role in lysosomal adaptation to starvation by activating the Ca2+-dependent phosphatase calcineurin, that de-phosphorylates and activates (Medina DL, et al, 2015). Most importantly, we also showed that TFEB over-expression promotes cellular clearance in different lysosomal storage disorders (LSDs) by inducing lysosomal exocytosis, a process that requires TRPML1-dependent Ca2+ release (Medina DL, et al, 2011). Interestingly, mutations in TRPML1 cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by psychomotor retardation and achlorhydria. The symptoms typically manifest in the first years of life and there is still no cure to treat patients affected by this disorder. At the cellular level MLIV-affected cells show accumulation of aberrant lysosomes filled with auto-fluorescent lipid material, including cholesterol. In addition, lysosomal dysfunction results in a partial block of autophagy while that TRPML1 over-expression promotes autophagy flux (Medina et al, 2015). Interestingly, accumulation of lipids as well as disturbed lysosomal Ca2+ homeostasis have been described in different LSDs and in some cases have been proposed to involve the lysosomal Ca2+ channel TRPML1, suggesting that TRPML1 impairment may be part of the pathogenic mechanisms in many other LSDs.
Our laboratory is now focused on; 1) the study of TRPML1 activation upon nutrient deprivation; 2) the role of TRPML1 and lysosomal calcium signalling in autophagy, and 3) the identification of TRPML1 interactors involved on lysosomal function and signalling. To study these important aspects of lysosomal biology we are utilizing molecular and cellular biology, High Content Imaging, and OMIC approaches. The final goal of our research is the discovery of compounds activating TRPML1-dependent pathways, and use these compounds to treat human diseases characterized by lysosomal/autophagic dysfunction.
High Content Screening Facility
Approximately 6,000 human genetic disorders are known to exist. According to the OMIM database, the responsible gene for over 30% of the currently known genetic diseasess has been identified. While molecular mechanisms are becoming increasingly recognizable thanks to persistent research efforts, therapeutic treatments have yet to see such progress. Since each genetic disease is particular in its rarity, genetic disorders as a whole have been, until very recently, neglected by the profit-driven structure of the system for drug development. As a result, the number of genetic diseases left untreated is still overwhelming. Thus, it is necessary to develop and implement a non-profit-driven process in order to discover effective treatments for genetic diseases.
Cell-based High Content Screening (HCS) has rapidly gained relevance, as it is being used in almost all of the pre-clinical steps of the drug discovery process. HCS is an analysis tool used to acquire, manage, and search multi-parametric information regarding the composite phenotype of cells. Thus HCS is important becaus eit allows for the simultaneous analysis of the impact of a given perturbation (gene modification or exposure to a drug) on such composite phenotypes. For these reasons, HCS is used during secondary and tertiary screening procedures to support hit-to-lead processes and mode-of-action studies, but also as a tool for primary screening in which thousands of compounds may be identified.
The HCS Facility at TIGEM-Pozzuoli has been tremendously upgraded with the integration of two high content microscopes (Opera and Operetta) with robotic plate-handlers, a liquid handling station (Zephyr) and a plate washer. This configuration significantly increases the throughput procedures executed in the facility. In addition we have a dedicated a cell culture room and a liquid handling station (StartLet Hamilton) to manipulation of compounds, siRNAs and cell lines, making our HCS facility suitable for DNA/RNAi-based high content screening. The volume of images and datapoints generated by the HCS Facility requires the HCS staff to collaborate with the Informatics and Bioinformatics TIGEM cores.
The main activities of the HCS Facility are
- Generation, development, validation and screening of disease cellular models of genetic diseases
- Identification of correctors for genetic diseases (small molecule libraries).
- Identification of drug targets for genetic diseases (druggable genome siRNA library).
- Identification of molecular pathways controlling specific cell functions (siRNA libraries).
- Supporting TIGEM researchers in the developing and realization of screening projects
Specific (HC)-cell based assays:
- Nuclear translocation assay
- Lysosomal morphometrics
- Lysosomal clearance
- Cell cycle (DNA content)
- Lysosomal membrane permeabilization (LMP)
- mTOR activity assay
- Focus small molecules libraries (around 1500 compounds; FDA compounds, kinase and phosphatase inhibitors, nuclear receptor ligands)
- siRNA libraries (11.000 genes, including druggable genome)
- microRNAs (1500)
StartLet liquid handler
Zephyr liquid handler
Multidrops Plate washers
Specialized cell culture room
Moskot M, Montefusco S, Jakobkiewicz-Banecka J, Mozolewski P, Wegrzyn A, Wegrzyn G, Medina DL, Ballabio A, Gabig-Ciminska M* (2014). The Phytoestrogen Genistein Modulates Lysosomal Metabolism and Transcription Factor EB (TFEB) Activation. J Biol Chem. 289(24):17054-17069. doi: 10.1074/jbc.M114.555300.
Medina DL, Di Paola S, Peluso I, Armani A, De Stefani D, Venditti R, Montefusco S, Scotto-Rosato A, Prezioso C, Forrester A, Settembre C, Wang W, Gao Q, Xu H, Sandri M, Rizzuto R, De Matteis MA, Ballabio A (2015). Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. Nat Cell Biol. 17(3):288-299
Song JX, Sun YR, Peluso I, Zeng Y, Yu X, Lu JH, Xu Z, Wang MZ, Liu LF, Huang YY, Chen LL, Durairajan SS, Zhang HJ, Zhou B, Zhang HQ, Lu A, Ballabio A, Medina DL*, Guo Z*, Li M*. A novel curcumin analog binds to and activates TFEB in vitro and in vivo independent of MTOR inhibition. Autophagy. 2016 Aug 2;12(8):1372-89. doi: 10.1080/15548627.2016.1179404. PubMed PMID: 27172265; PubMed Central PMCID: PMC4968239.
Medina DL, Fraldi A, Bouchè V, Annunziata F, Mansueto G, Spampanato C, Puri C, Pignata A, Martina JA, Sardiello M, Polischuk R, Puertollano R, Ballabio A (2011). Transcriptional activation of lysosomal exocytosis promotes cellular clearance. Dev Cell. 21: 421-430. doi: 10.1016/j.devcel.2011.07.016.
Settembre C, Medina DL(2015). TFEB and the CLEAR network. In F. Platt, & N. Platt (Eds.), Lysosomes and lysosomal diseases. 126: 45-62. doi:10.1016/bs.mcb.2014.11.011.
The HCS Facility also conducts research focused on the discovery of new ‘druggable’ targets and the development of pharmacological strategies to treat rare genetic diseases by using cell biology approaches and high-content imaging technology. To identify new ‘druggable’ targets we rely on RNAi-based screenings. We have developed automated protocols to transfect cells with siRNA oligonucleotides using the Hamilton Startlet liquid handling station. We house various molecule libraries, but our expanded version of an RNAi-collection is especially important for targeting the druggable genome. Indeed, this approach is particularly useful in helping us understand the signal transduction pathways involved in the pathogenesis of rare diseases.
Using such strategies demands relevant cellular models of genetic diseases. Thus we are using CRISPR technology, siRNA-mediated silencing and overexpression of fluorescent reporter genes to generate suitable cellular models and multi-parametric read-outs to be used in several screening projects.