A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha-synuclein to lysosomes
Description
A key question in PD research relates to the mechanism by which alpha-synuclein (α-syn) fibrils enter cells. It is generally thought that α-syn preformed fibrils (PFF), a toxic form of α-syn, are trafficked to lysosomes through the endosomal system over the course of hours, although many conflicting results exist in the literature. We believe that some of the conflicting evidence arises from examining α-syn internalization at longer time courses and not immediately after its addition to cells. We also believe that dynamin inhibition, employed in many studies to inhibit PFF internalization, should not be treated as synonymous with inhibition of clathrin-mediated endocytosis (CME), as the role of dynamin spans beyond CME and is involved in multiple endocytic pathways and actin polymerization. We thus sought to examine PFF endocytosis immediately after its addition to cells. Remarkably, PFF are internalized rapidly and appear in lysosomes within 2 min, completely bypassing conventional endosomal trafficking pathways. We confirmed this result in multiple cell lines, primary human cells, and neurons derived from human induced pluripotent stem cells (hiPSC). The internalization is not dependent on clathrin but instead utilizes macropinocytosis. All the raw data collected in our manuscript, has been organized and included in the .xlsx file attached. Additionally, the statistical calculations computed, with the use of Prism (graphpad.com) have also been included.
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Classical endocytic assays were adapted to look at PFF internalization. In examining PFF uptake, one must factor in the random nature of this protein, and therefore, the results are hardly uniform, in terms of the amount of internalization per cell, even within the same coverslip. PFF tends to attach very quickly and be internalized by cells directly after its addition to cells. This usually means some regions of the coverslips will contain cells with many PFF puncta, while other regions will be devoid of any PFF; therefore, it is important to examine the entire coverslip under the microscope in order to find cells that have internalized PFF at these early time points. At later timepoints, this variability is lessoned, because all cells have had some level of PFF uptake. Most importantly, PFF needs to be added directly to the coverslip/sample you want to examine and cannot be diluted in large amounts of media and then administered to cells. The latter would result in some samples showing a lot of uptake, and others showing no uptake at all, as the tendency for PFF to aggregate is extremely high. Following the establishing of the PFF endocytic assays, we realized the negative effects of fixation and permeabilization on PFF fluorescence. Hence, we avoided immunofluorescence as much as we possibly could. As an alternative, PFF uptake was first quantified in cells transiently expressing LAMP1-RFP, starting as early as 2 minutes. Transferrin was used as a marker of early/recycling endosomes, and was added to cells along side PFF to determine the PFF trafficking pathway. Once the assay was established in U2OS and HeLa, we examined PFF uptake in more relevant cell types, such as iPSC derived human dopaminergic neurons, primary human astrocytes, and human glioblastoma cell lines. We found that U2OS and astrocytes take up large amounts of PFF, and were one of the few cell lines which internalized enough PFF to withstand the loss of PFF fluorescence following permeabilization; hence, most of our immunofluorescence experiments were conducted in these two cell types. Lastly, it is important to note that a highly sensitive confocal microscope needs to be used to detect PFF fluorescence at such early time points. We have examined our samples using small benchtop fluorescent microscopes with a 10-20X lens, and the PFF fluorescence cannot be seen at 2 min or even 10 min. Conventional high content screening of our early time point experiments is also not possible. Highly sensitive detectors, high magnification, and fluorescent tags with a high quantum yield such as Alexa Fluor 488 (ThermoFisher), are required for spotting PFF uptake. Please refer to the paper, under the same title, or visit preprint: https://doi.org/10.1101/2022.01.06.475207 for full methodology.