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 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.
Highlights
Morphological alterations of subcellular organelles from the endosomal pathway have been extensively described in Alzheimer’s disease (AD) and Down syndrome (DS), notably an abnormal increase in the size of early endosomes in pyramidal neurons of the cortex [10].Such alteration of the morphology of endosomes is still considered as the earliest neuropathological hallmark of AD since it occurs in the neocortex of patients with sporadic AD, in Familial early onset AD (FAD) with mutations in the gene encoding the Amyloid Precursor Protein (APP), and in DS individuals carrying a trisomy for human chromosome 21 (HSA21) before amyloid peptides deposition [9, 14]
* Correspondence: marie-claude.potier@upmc.fr 1Paris Brain Institute (ICM), CNRS UMR7225, INSERM U1127, Sorbonne Université, Hôpital de la Pitié-Salpêtrière, Paris, France Full list of author information is available at the end of the article
Ultrastructure of Endosome Antigen 1 (EEA1)-positive early endosomes in Lymphoblastoid cell line (LCL) from individuals with DS Using confocal microscopy, we have previously shown that LCLs from individuals with DS contain enlarged EEA1-and Rab5-positive puncta as compared to euploid individuals [20]
Summary
Morphological alterations of subcellular organelles from the endosomal pathway have been extensively described in Alzheimer’s disease (AD) and Down syndrome (DS), notably an abnormal increase in the size of early endosomes in pyramidal neurons of the cortex [10]. Such alteration of the morphology of endosomes is still considered as the earliest neuropathological hallmark of AD since it occurs in the neocortex of patients with sporadic AD, in Familial early onset AD (FAD) with mutations in the gene encoding the Amyloid Precursor Protein (APP), and in DS individuals carrying a trisomy for human chromosome 21 (HSA21) before amyloid peptides deposition [9, 14]. Previous studies showed endosomal enlargement in the brain of mouse models of FAD with mutations in the APP gene [16] and in Ts65Dn mice [12], the best-characterized and most widely used mouse model of DS
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