Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

Abstract

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 MVBdependent 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.

Stress Responses in Down Syndrome Neurodegeneration: State of the Art and Therapeutic Molecules

“…Endosomal pathology has been also shown in human DS neurons, in neurons of DS mouse models, and in human DS fibroblasts [ 188 , 189 , 190 , 191 , 192 ]. Several evidence and emerging hypotheses suggest that triplicated genes, such as ITSN1 and SYNJ1, are implicated in triggering the abnormalities of endolysosomal pathway [ 193 ].…”

Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21)

“…In agreement, other reports have noted a ~18% increase in the size of EEA1+ vesicles without significant differences in the number of this type of vesicles. Enlargement of lysosomes in fibroblasts from individuals with trisomy 21 has also been reported 45,47 . Of note, the average size of LAMP1+ vesicles in fixed cells with trisomy 21 was smaller.…”

“…Alterations in the morphology of endosomes have been described in primary fibroblasts, neurons, peripheral blood mononuclear cells, and lymphoblastoid cell lines from individuals with Down syndrome 36,37,[43][44][45] . Our comparative morphological analysis of IgG+ vesicles and subcellular compartments in fibroblasts with and without trisomy 21 revealed enlargement of subcellular organelles from the endosomal pathway, resembling the "traffic jam" previously described for DS and Alzheimer's disease 45,46 . We found that trisomic cells have larger EEA1+ endosomes (~12%), Rab11+ endosomes (~24%), and lysotracker-stained lysosomes (28%) than diploid cells (Figure 2).…”

“…Several studies implemented receptor-mediated uptake approaches to examine endosomal defects in trisomic cells 37,45,47 . There is limited information describing IgG traffic following internalization by fluid phase pinocytosis in the context of trisomy 21 21,49 .…”

“…Figures 1 and 5). In diploid fibroblasts overexpressing APP, the number and size of intracellular IgG vesicles remained unaltered, which suggests that other factors contribute to morphological alterations in trisomic cells in addition to increased APP expression 44,45 .…”