This project focused on lysosomes, an organelle with critical functions in cells. By contributing to a better understanding of the lysosome, the project aims to have a long-term 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 (LSDs) to age-related neurodegenerative disorders.
Like most genetic diseases, LSDs present a wide range of clinical phenotypes, even in siblings sharing a common mutation. Based on the hypothesis that modifier genes play a role in such phenotypic variability, the first objective of this project was to identify and characterize genetic modifiers of lysosomal function (WP1). Genomic DNA from patients was analyzed by exome sequencing, and polymorphisms that segregated with disease severity were identified. Additionally, a systems genetics approach was used to identify putative modifier genes/networks in 27 strains of mice, revealing new genes, networks, and biological processes (
https://doi.org/10.1016/j.bbrep.2021.101105(odnośnik otworzy się w nowym oknie)).
Metal imbalances have been described in LSDs, and the second objective of this project was to investigate whether therapies that target metal imbalances can be beneficial for treating LSDs (WP2). WP2 further included studies on the cellular and molecular mechanism of action of acetyl-DL-leucine. A known iron chelator was modified and covalently attached to a cyclodextrin in order to make a hydrid drug for potential NPC treatment. Assays in NPC cell lines showed iron/copper chelation (
https://doi.org/10.1002/cmdc.201900334(odnośnik otworzy się w nowym oknie);
https://doi.org/10.1007/s10534-019-00185-5(odnośnik otworzy się w nowym oknie)). Moreover, pre-clinical and clinical studies revealed a neuroprotective effect of acetyl-leucine and underlying mechanisms of action in lysosomal storage diseases (see publications:
https://doi.org/10.3390/jcm9041050(odnośnik otworzy się w nowym oknie);
https://doi.org/10.1093/braincomms/fcaa148(odnośnik otworzy się w nowym oknie)).
The third objective of this project was to investigate the cross-talk between lysosomal function, signalling pathways and gene expression regulation (WP3). A link between c-Abl and TFEB was uncovered. c-Abl was shown to act as a TFEB regulator that mediates its tyrosine phosphorylation, and the inhibition of c-Abl activates TFEB promoting cholesterol clearance in NPC models (
https://doi.org/10.1016/j.isci.2020.101691(odnośnik otworzy się w nowym oknie)). Further work on RIPK3 showed that c-Abl signaling is a new upstream pathway that activates RIPK3 and that its inhibition is an attractive therapeutic approach for the treatment of Gaucher disease (DOI: 10.1016/j.bbadis.2021.166089).
Using multiple biophysical methodologies, this project led to the discovery that the fluidity of the cell membranes is altered in models of NPC and that these changes are in part caused by sphingosine (doi.org/10.1016/j.bbalip.2021.158944). Moreover, cholesteryl hemiazelate was identified by lipidomics as one of the molecules involved in the etiology of irreversible lysosome dysfunction culminating with lipidosis (
https://doi.org/10.1101/2021.01.05.422575(odnośnik otworzy się w nowym oknie)).
Finally, based on recent evidence suggesting that lysosomes act as signalling organelles that influence nuclear transcription and that lipids produced in the lysosome move to the nucleus altering gene expression, WP3 aimed to investigate whether alternative splicing is targeted by lysosome-mediated signalling pathways. An RNA-seq analysis performed in primary fibroblasts derived from GD, NPC and NPA patients, revealed for the first time multiple alternative splicing events associated specifically with each disease. Results from this WP further revealed that perturbations in lysosomal glycosphingolipid metabolism stimulate cell division, which may be implicated in control of neurogenic niches. Mis-regulated alternative splicing of mRNAs encoding synaptic proteins was also observed, raising the possibility that perturbations synaptic functions may contribute to the initial stages of the neuropathological processes underlying Gaucher and Parkinson’s disease (three research articles reporting these observations are in preparation).
