Periodic Reporting for period 1 - LySyT (Understanding the role of lysosomes in the intercellular TNT-mediated spreading of α-synuclein and the impact of lysosomal dysfunction)
Período documentado: 2020-12-01 hasta 2022-11-30
Although neurodegenerative diseases (NDs) are one of the top 10 global causes of death, our knowledge about the basic molecular mechanisms underlying their pathogenesis is still lacking. The accumulation of misfolded protein aggregates in affected brain regions is a common hallmark shared by several NDs. In Parkinson’s disease (PD), misfolded alpha-synuclein (α-syn) proteins accumulate within neurons in the so-called Lewy bodies. Substantial findings support the capability of misfolded α-syn proteins to propagate in cellular and in animal models by transferring between cells and by inducing the aggregation of endogenous proteins (seeding activity). Lysosomes are responsible for the degradation of misfolded aggregates and their dysfunction is a common trait of NDs; however, the mechanisms leading to lysosomal damage and how this damage contributes to neurodegeneration is not clear. In this project we applied the state-of-the-art imaging approaches both in live and fixed conditions (e.g. confocal live-cell imaging microscopy, super resolution microscopy, and correlative electron microscopy) together with biochemistry and molecular biology to test our ground-breaking hypothesis that dysfunctional lysosomes are transferred through Tunneling nanotubes (TNTs), membranous bridges connecting distant cells, and contribute to the propagation of the pathology. It has been previously shown that α-syn fibrils are transferred through TNTs between neurons while inside lysosomes, and that the fibrils induced the misfolding and aggregation of the normal soluble protein after their transfer. Here, the LySyT project focused on outstanding questions: i) how lysosomes enter and move inside TNS; ii) how α-syn fibrils, while inside lysosomes, could seed the misfolding of soluble α-syn in recipient cells; iii) where seeding occurs inside the cell and the role of lysosomes in this event; and iv) the fate of α-syn-loaded lysosomes. This project had high translational application, as it aimed at understanding of the mechanisms of propagation of the α-syn pathology and the effects on neurons, which are essential step to identify suitable therapeutic targets for PD.
Lysosomal dysfunction is also a common feature of Lysosomal Storage Diseases (LSDs), a group of about 50 rare, inherited metabolic disorders caused by the defective function of a specific lysosomal enzyme. Classically, LSDs are classified in diverse forms encompassing a wide spectrum of clinical phenotypes and the most severe forms of LSDs affect the nervous system. With the aim to better elucidate the pathogenesis of LSDs and to highlight possible similarities with NDs, the LySyT project also studied the possible role of TNTs in the lysosomal pathology of LSDs.
*Image name: The lysosome: an ally in spreading Parkinson’s disease. Image description: 3D rendering of a lysosome (grey) facilitating the formation of new α-syn aggregates (green) induced by the pre-existing α-syn aggregates (red).
Lysosomal dysfunction is also a common feature of Lysosomal Storage Diseases (LSDs), a group of about 50 rare, inherited metabolic disorders caused by the defective function of a specific lysosomal enzyme. Classically, LSDs are classified in diverse forms encompassing a wide spectrum of clinical phenotypes and the most severe forms of LSDs affect the nervous system. With the aim to better elucidate the pathogenesis of LSDs and to highlight possible similarities with NDs, the LySyT project also studied the possible role of TNTs in the lysosomal pathology of LSDs.
*Image name: The lysosome: an ally in spreading Parkinson’s disease. Image description: 3D rendering of a lysosome (grey) facilitating the formation of new α-syn aggregates (green) induced by the pre-existing α-syn aggregates (red).
The LySyT project shed new light on the role of lysosomes in the propagation of α-syn pathology through TNTs. First, we showed that α-syn-loaded lysosomes localize more at the cell periphery compared to control lysosomes. In addition, we found that peripheral α-syn-loaded lysosomes do not undergo lysosomal exocytosis and they are more prone to be transferred to the acceptor cells through TNTs instead. Concerning the transfer of lysosomes through TNTs, we investigated several candidates to possibly find the motor protein responsible for the movement of lysosomes along the actin filaments composing the TNTs.
We also showed that in our model, lysosomes containing α-syn undergo to Lysosomal Membrane Permeabilization (LMP) both in donor cells pre-loaded with the fibrils and acceptor cells that received the fibrils after the transfer from the donors. Our preliminary results also suggest that cells challenged with α-syn fibrils respond first recruiting a rescue machinery at the damaged lysosomes as an attempt followed by the incapacity to counteract the LMP event. Interestingly, we found that new α-syn aggregates, co-localizing with α-syn fibrils, localize mostly inside the lysosomes or at the lysosomal membrane, both in donor cells loaded with α-syn fibrils and in acceptor cells that received the fibrils from the pre-loaded donors. These results reveal the role of lysosomes acting as a hub facilitating the formation of new aggregates (i.e. seeding).
Concerning the fate and the functionality of α-syn-loaded lysosomes, we found that they are less acidic, less functional, and enlarged compared to control lysosomes. We did not find any change in lysosomal biogenesis since the total number of lysosomes is comparable to that of control cells. Regarding autophagy, our results show that α-syn-loaded lysosomes do not undergo lysophagy. In addition, we optimized a previously described protocol to purify α-syn-loaded lysosomes to analyze their protein composition by mass spectrometry.
Concerning the possible role of TNTs in the lysosomal pathology of LSDs, by using an artificial model of LSDs (i.e. neuronal cells loaded with sucrose) we found an increased percentage of TNT-connected cells compared to control cells. As in the α-syn-loaded cells, in sucrose-loaded cells we found TFEB nuclear translocation not followed by an increased lysosomal biogenesis although our preliminary results indicate an enlargement of sucrose-loaded lysosomes compared to control lysosomes. In addition, sucrose-loaded lysosomes are less functional compared to control lysosomes and the autophagy pathway is likely not induced. Our preliminary results also indicate that sucrose-loaded cells are more prone to transfer vesicles towards acceptor cells compared to control donor cells, suggesting a possible increase in lysosomes’ transfer as well.
The results of the LySyt project have been disseminated during international conferences such as the “Club Exocytose-Endocytose” meeting, the “Cell la vie” conference organized by the French Society for Cell Biology (SBCF), the “AD/PD 2022 Alzheimer's & Parkinson's Diseases Conference” as well as during the annual retreats of the Cell Biology and Infection Department of the Institut Pasteur. One manuscript describing the mechanism of α-syn lysosomal escape in the context of the protein pathology spreading and the effects of α-syn exposure on lysosome function has been published on the open-access journal PLOS Biology. Outreach activities have been carried out to communicate the research of the LySyT project to the public such as workshops organized by the European Commission (Science is wonderful) and by the European association Native Scientists and a public meeting organized by the association France Parkinson.
We also showed that in our model, lysosomes containing α-syn undergo to Lysosomal Membrane Permeabilization (LMP) both in donor cells pre-loaded with the fibrils and acceptor cells that received the fibrils after the transfer from the donors. Our preliminary results also suggest that cells challenged with α-syn fibrils respond first recruiting a rescue machinery at the damaged lysosomes as an attempt followed by the incapacity to counteract the LMP event. Interestingly, we found that new α-syn aggregates, co-localizing with α-syn fibrils, localize mostly inside the lysosomes or at the lysosomal membrane, both in donor cells loaded with α-syn fibrils and in acceptor cells that received the fibrils from the pre-loaded donors. These results reveal the role of lysosomes acting as a hub facilitating the formation of new aggregates (i.e. seeding).
Concerning the fate and the functionality of α-syn-loaded lysosomes, we found that they are less acidic, less functional, and enlarged compared to control lysosomes. We did not find any change in lysosomal biogenesis since the total number of lysosomes is comparable to that of control cells. Regarding autophagy, our results show that α-syn-loaded lysosomes do not undergo lysophagy. In addition, we optimized a previously described protocol to purify α-syn-loaded lysosomes to analyze their protein composition by mass spectrometry.
Concerning the possible role of TNTs in the lysosomal pathology of LSDs, by using an artificial model of LSDs (i.e. neuronal cells loaded with sucrose) we found an increased percentage of TNT-connected cells compared to control cells. As in the α-syn-loaded cells, in sucrose-loaded cells we found TFEB nuclear translocation not followed by an increased lysosomal biogenesis although our preliminary results indicate an enlargement of sucrose-loaded lysosomes compared to control lysosomes. In addition, sucrose-loaded lysosomes are less functional compared to control lysosomes and the autophagy pathway is likely not induced. Our preliminary results also indicate that sucrose-loaded cells are more prone to transfer vesicles towards acceptor cells compared to control donor cells, suggesting a possible increase in lysosomes’ transfer as well.
The results of the LySyt project have been disseminated during international conferences such as the “Club Exocytose-Endocytose” meeting, the “Cell la vie” conference organized by the French Society for Cell Biology (SBCF), the “AD/PD 2022 Alzheimer's & Parkinson's Diseases Conference” as well as during the annual retreats of the Cell Biology and Infection Department of the Institut Pasteur. One manuscript describing the mechanism of α-syn lysosomal escape in the context of the protein pathology spreading and the effects of α-syn exposure on lysosome function has been published on the open-access journal PLOS Biology. Outreach activities have been carried out to communicate the research of the LySyT project to the public such as workshops organized by the European Commission (Science is wonderful) and by the European association Native Scientists and a public meeting organized by the association France Parkinson.
The LySyT project pointed out a new role for the lysosomes in the spreading of α-syn pathology through TNTs, opening new perspectives in identifying new therapeutic targets for PD. Moreover, it will be interesting to evaluate a similar pathogenic role for the lysosomes in other NDs and in LSDs, as suggest our preliminary results obtained in the sucrose model.