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Genetic and Small Molecule Modifiers of Lysosomal Function

Periodic Reporting for period 1 - LysoMod (Genetic and Small Molecule Modifiers of Lysosomal Function)

Reporting period: 2017-03-01 to 2019-02-28

Based on the critical role that lysosomes play in cells, the concept behind this project is that a better understanding of lysosomal function will have a major impact on human health, fostering the development of new strategies that will help improve the quality of life of people affected by a variety of diseases, ranging from lysosomal storage diseases to age-related neurodegenerative disorders.
The overall objectives of the project are:
1) to identify and characterize genetic modifiers of lysosomal function, which may represent potential new targets for personalized therapeutic approaches;
2) to unravel the mechanism of action of small molecule modifiers of lysosomal function;
3) to investigate the cross-talk between lysosomal function, signaling pathways and gene expression regulation.
Previous work from Andrés Klein in the Futerman lab identified putative single nucleotide polymorphisms (SNPs) that may be involved in determining Gaucher disease severity in a mouse model. In collaboration with the Futerman lab, Christian Ramos (GenoMed) found that the majority of these SNPs are located in noncoding regions of the genome, namely in introns. To experimentally determine how these SNPs may affect gene expression in neuronal cells, protocols have been optimized to differentiate neurons in vitro from either mouse embryonic stem cells or neural progenitors. RNA obtained from neurons differentiated from different mouse strains will be analyzed by RNA-seq. Further work performed during secondments from GenoMed to the Futerman lab aimed to identify how lipid accumulation in cells from Gaucher patients with distinct genotypes affects the biophysical properties of biological membranes.
The Zanlungo lab has analyzed by exome sequencing a cohort of 10 homozygous Chilean patients with Niemann-Pick type B disease presenting phenotypic variability. Identification of polymorphisms that segregate with the degree of severity of liver disease was carried out in collaboration with Andres Klein.
A collaboration between the Klein and Platt labs has started to characterize a family of NPC patients where siblings show different disease severity. Using patient derived fibroblasts, a multi-omics approach was designed to identify modifier genes/pathways. The strategy includes exome sequencing, proteomic studies, cell biology characterization (lysosomal and mitochondrial markers), electron microscopy, and lipid analysis.

The Platt lab studied the effects of acetyl-DL-leucine and acetyl-D- leucine and acetyl-L-leucine in a mouse model of NPC1 (Npc1-/-) disease. They have found that all three analogues show benefit in NPC1 mice. The NPC1 mice live slightly longer, have slowed disease progression and improved motor function. Purkinje cell survival is also enhanced by all three analogues and glycosphingolipid storage levels reduced in the brain and peripheral organs. Building on these findings we are investigating mechanism of action collaborating with LysoMod groups to identify protein binding partners of biotinylated analogues (made by the Spencer lab) with proteomics of binding proteins (performed in Chronos in collaboration with the Petrescu lab), and transcriptomic analysis (performed in GenoMed in collaboration with the Carmo-Fonseca lab).

A collaboration between Intrabio and the Futerman lab was established to study microglial gene expression in murine models of lysosomal storage disease. In brief, the rationale is to undertake RNA-sequencing of primary microglia isolated from different murine models with a view to furthering our understanding of the biological contributions of these cells to CNS disease, identification of both common signatures and disease-specific ones and new therapeutic targets.
The Zanlungo lab previously found the ABL tyrosine kinase is aberrantly active in NPC, and they demonstrated that several ABL inhibitors promote cellular clearance of cholesterol in both in vitro and in vivo NPC models. In collaboration with the Platt laboratory, inmmunofluorescence analysis of lysosomes in Cells treated with the ABL inhibitor Imatinib and a siRNA against ABL were studied using cell biology assays (in collaboration with the Platt lab) and RNA sequencing (in collaboration with the Carmo-Fonseca lab).
Finally, a new collaboration was established between the Srebrow and Carmo-Fonseca groups to study the coupling between autophagy and gene expression regulation in Dengue infected cells.
Disorders linked to lysosomal dysfunction still represent a large and growing unmet medical need. By contributing to a better understanding of lysosomal function this project has the potential to foster the development of new strategies that will help improve the quality of life of people affected by either LSDs or age-related neurodegenerative disorders. Until the end of the project, we expect to identify new molecules that are critical for lysosomal function and determine whether manipulating their expression and/or activity represents an opportunity to clear lysosomal storage defects. Moreover, the project is effectively enhancing the potential and future career perspectives of the staff members, as well as developing new research collaborations, achieving transfer of knowledge between participating organizations and contributing to improving research and innovation potential at the European and global levels. Indeed, the LysoMod consortium is providing young researchers with high-level training in innovative approaches for exploring biological systems, preparing the next generation of researchers for careers either in private and public health sectors. Building on close training collaborations between the participating institutions, the LysoMod project is creating a network of synergies and exchanges of ESRs. The project has already promoted joint research initiatives and the development of new collaborations. Moreover, the integration of companies in the project is contributing to the training of ESRs with skills that will be critically required for further growth in the intersection of the public and private sector.