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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 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.

SUBMITTER: Botte A

PROVIDER: S-EPMC7315513 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome.

Botté Alexandra A Lainé Jeanne J Xicota Laura L Heiligenstein Xavier X Fontaine Gaëlle G Kasri Amal A Rivals Isabelle I Goh Pollyanna P Faklaris Orestis O Cossec Jack-Christophe JC Morel Etienne E Rebillat Anne-Sophie AS Nizetic Dean D Raposo Graça G Potier Marie-Claude MC

Acta neuropathologica communications 20200624 1

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 Endoso ...[more]

PMID: 32580751

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Project description:Phosphatidylinositol-3-phosphate (PI3P), a major identity tag of early endosomes (EEs), provides a platform for the recruitment of numerous cellular proteins containing an FYVE or PX domain that is required for PI3P-dependent maturation of EEs. Most of the PI3P in EEs is generated by the activity of Vps34, a catalytic component of class III phosphatidylinositol-3-phosphate kinase (PI3Ks) complex. In this study, we analyzed the role of Vps34-derived PI3P in the EE recycling circuit of unperturbed cells using VPS34-IN1 (IN1), a highly specific inhibitor of Vps34. IN1-mediated PI3P depletion resulted in the rapid dissociation of recombinant FYVE- and PX-containing PI3P-binding modules and endogenous PI3P-binding proteins, including EEA1 and EE sorting nexins. IN1 treatment triggered the rapid restructuring of EEs into a PI3P-independent functional configuration, and after IN1 washout, EEs were rapidly restored to a PI3P-dependent functional configuration. Analysis of the PI3P-independent configuration showed that the Vps34-derived PI3P is not essential for the pre-EE-associated functions and the fast recycling loop of the EE recycling circuit but contributes to EE maturation toward the degradation circuit, as previously shown in Vps34 knockout and knockdown studies. However, our study shows that Vps34-derived PI3P is also essential for the establishment of the Rab11a-dependent pathway, including recycling cargo sorting in this pathway and membrane flux from EEs to the pericentriolar endosomal recycling compartment (ERC). Rab11a endosomes of PI3P-depleted cells expanded and vacuolized outside the pericentriolar area without the acquisition of internalized transferrin (Tf). These endosomes had high levels of FIP5 and low levels of FIP3, suggesting that their maturation was arrested before the acquisition of FIP3. Consequently, Tf-loaded-, Rab11a/FIP5-, and Rab8a-positive endosomes disappeared from the pericentriolar area, implying that PI3P-associated functions are essential for ERC biogenesis. ERC loss was rapidly reversed after IN1 washout, which coincided with the restoration of FIP3 recruitment to Rab11a-positive endosomes and their dynein-dependent migration to the cell center. Thus, our study shows that Vps34-derived PI3P is indispensable in the recycling circuit to maintain the slow recycling pathway and biogenesis of the ERC.

Dataset Information

Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome.

Publications

Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome.

Similar Datasets

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