Periodic Reporting for period 2 - LysoMod (Genetic and Small Molecule Modifiers of Lysosomal Function)
Okres sprawozdawczy: 2019-03-01 do 2022-08-31
The LysoMod project contributed innovations in the area of personalized medicine for disorders linked to lysosomal dysfunction (both rare and common) by implementing an international staff-exchange program between highly complementary and multidisciplinary academic experts and non-academic partners. Research over the past decades has clearly shown that as new aspects of lysosomal biology are revealed, new therapeutic strategies can be devised, in most cases based on the manipulation of specific molecular pathways. In summary, the LysoMod project has 1) identified genetic variation that may explain why different patients with the same causative mutation (including siblings) present a wide range of clinical symptoms; 2) demonstrated that acetyl-leucine (a derivative of amino acid leucine) exerts a neuroprotective effect in mouse models and in patients with lysosomal diseases; and 3) uncovered cellular components and signaling pathways involved in lysosomal dysfunction that may be targets for the development of new therapeutic approaches.
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).
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; https://doi.org/10.1007/s10534-019-00185-5). 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; https://doi.org/10.1093/braincomms/fcaa148).
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). 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).
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).
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).
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; https://doi.org/10.1007/s10534-019-00185-5). 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; https://doi.org/10.1093/braincomms/fcaa148).
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). 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).
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).
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 rare lysosomal diseases or common age-related neurodegenerative disorders. The consortium has identified new molecules that are critical for lysosomal function and shown that their manipulation represents an opportunity for treatment. The project has effectively enhanced the potential and future career perspectives of the staff members, and developed new research collaborations, achieving transfer of knowledge between participating organizations and contributing to improving research and innovation potential at the European and global levels. The consortium has provided 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, it has created a network of synergies and exchanges of ESRs. The integration of companies in the project has contributed to the training of ESRs with skills that will be critically required for further growth in the intersection of the public and private sector. Notably, the project promoted joint research initiatives that culminated with the approval by the European Commission of a new follow-up project (RiboMed GA857119) that is coordinated by M. Carmo-Fonseca and involves two additional partners of the LysoMod consortium.