Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

Acta Neuropathol Commun. 2020 Jun 24;8(1):89. doi: 10.1186/s40478-020-00956-z.

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.

Keywords: Alzheimer’s disease; Down syndrome; Early endosomes; Electron microscopy; Endocytosis; Super-resolution microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Down Syndrome / metabolism
  • Down Syndrome / pathology*
  • Endosomes / metabolism*
  • Endosomes / ultrastructure*
  • Fibroblasts / metabolism
  • Fibroblasts / ultrastructure*
  • Humans
  • Induced Pluripotent Stem Cells
  • Mice
  • Microscopy, Confocal
  • Microscopy, Electron, Transmission
  • Tissue Fixation
  • Vitrification