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.
