Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

Enlarged early endosomes have been visualized in Alzheimer's disease (AD) and Down syndrome (DS) using conventional confocal microscopy at a resolution corresponding to endosomal size (hundreds of nm). In order to overtake the diffraction limit, we used super-resolution structured illumination microscopy (SR-SIM) and transmission electron microscopies (TEM) to analyze the early endosomal compartment in DS.By immunofluorescence and confocal microscopy, we confirmed that the volume of Early Endosome Antigen 1 (EEA1)-positive puncta was 13-19% larger in fibroblasts and iPSC-derived neurons from individuals with DS, and in basal forebrain cholinergic neurons (BFCN) of the Ts65Dn mice modelling DS. However, EEA1-positive structures imaged by TEM or SR-SIM after chemical fixation had a normal size but appeared clustered. In order to disentangle these discrepancies, we imaged optimally preserved High Pressure Freezing (HPF)-vitrified DS fibroblasts by TEM and found that early endosomes were 75% denser but remained normal-sized.RNA sequencing of DS and euploid fibroblasts revealed a subgroup of differentially-expressed genes related to cargo sorting at multivesicular bodies (MVBs). We thus studied the dynamics of endocytosis, recycling and MVB-dependent degradation in DS fibroblasts. We found no change in endocytosis, increased recycling and delayed degradation, suggesting a "traffic jam" in the endosomal compartment.Finally, we show that the phosphoinositide PI (3) P, involved in early endosome fusion, is decreased in DS fibroblasts, unveiling a new mechanism for endosomal dysfunctions in DS and a target for pharmacotherapy.National Research Foundation (NRF)Published versionMarie-Claude Potier was supported by Fondation Vaincre Alzheimer (fellowship to AB), Fondation Jérôme Lejeune and Agence Nationale de laRecherche (ANR-10-IAIHU-06). Dean Nizetic was funded by Wellcome Trust (098330/Z/12/Z), NationalResearch Foundation Singapore (NMRC/CS-IRG/1438/2015), the WellcomeTrust Collaborative Award in Science 217199/Z/19/Z, and by the “Research Cooperability“ Programme of the Croatian Science Foundation (PZS-2019-02-4277). France-BioImaging infrastructure was supported by the French National Research Agency (ANR-10-INBS-04).