ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-β accumulation

Abstract

Intracellular amyloid-β (Aβ) accumulation is a key feature of early Alzheimer's disease and precedes the appearance of Aβ in extracellular plaques. Aβ is generated through proteolytic processing of amyloid precursor protein (APP), but the intracellular site of Aβ production is unclear. APP has been localized to multivesicular bodies (MVBs) where sorting of APP onto intraluminal vesicles (ILVs) could promote amyloidogenic processing, or reduce Aβ production or accumulation by sorting APP and processing products to lysosomes for degradation. Here, we show that APP localizes to the ILVs of a subset of MVBs that also traffic EGF receptor (EGFR), and that it is delivered to lysosomes for degradation. Depletion of the endosomal sorting complexes required for transport (ESCRT) components, Hrs (also known as Hgs) or Tsg101, inhibited targeting of APP to ILVs and the subsequent delivery to lysosomes, and led to increased intracellular Aβ accumulation. This was accompanied by dramatically decreased Aβ secretion. Thus, the early ESCRT machinery has a dual role in limiting intracellular Aβ accumulation through targeting of APP and processing products to the lysosome for degradation, and promoting Aβ secretion.

Keywords: Alzheimer's disease; Amyloid precursor protein; ESCRT; Multivesicular body.

Fig. 1.

APP localizes to the Golgi and ILVs of a subpopulation of MVBs. (A) Untransfected H4-UnT and H4-APP cells were stained for APP/Aβ (6E10; green), TGN46 (magenta) and DAPI (blue). Scale bar: 10 μm. (B) In ultrathin cryosections of H4-APP cells, 6E10 immunogold labelling is on a subset of MVBs (asterisks) and the Golgi, although some MVBs lack label (arrows). Scale bars: 100 nm. (C) Counting 6E10 gold particles per MVB (n=120) shows populations of 6E10 negative and positive MVBs (approximately four particles per MVB). (D) H4-UnT and H4-APP cells were western blotted with 6E10.

Fig. 2.

APP and Aβ localizes to the ILVs of a subpopulation of MVBs in hippocampus of Tg2576 mice. Ultrathin sections of hippocampus of Tg2576 mice were stained with the anti-APP/Aβ antibody 6E10 (A) or Aβ40-specific antibody G2-10 (B). Arrows show MVBs negative of gold labelling. Scale bars: 200 nm.

Fig. 3.

APP traffics through the same MVBs as EGFR. (A) H4-APP cells stimulated with EGF–Alexa-Fluor-488 (EGF-488; green) for 10 or 45 min were stained for APP/Aβ with 6E10 (red). Scale bar: 5 μm. (B) Quantification of APP colocalization with EGF-488. (C) H4-APP cells treated with leupeptin were stained with 6E10 (green), cathepsin D (red) and DAPI (blue). Scale bars: 10 μm. (D) Quantification of 6E10 colocalization with cathepsin D. All results are mean±s.d. (n=3 separate experiments). *P<0.05, ***P<0.005 (Student's t-test).

Fig. 4.

Redistribution of APP from ILVs to the limiting membrane of MVBs after Hrs depletion. (A) Cryosections of H4-APP and Hrs-depleted H4-APP cells (siHrs) were stained for APP/Aβ with 6E10 (upper two panels, cells fixed with 4% PFA; lower panel, cells fixed with 4% PFA and 0.2% glutaraldehyde). Scale bars: 200 nm. (B) Confocal microscopy reveals 6E10-positive endosomal rings following Hrs depletion (arrows). Scale bars: 5 μm.

Fig. 5.

Reduced lysosomal delivery after Hrs/Tsg101 depletion. (A) H4-APP cells depleted of Hrs (siHrs) or Tsg101 (siTsg101) were treated with or without leupeptin and stained with 6E10 (green) and LAMP1 (red). Scale bars: 5 μm. (B) Quantification of 6E10 colocalization with LAMP1 (using Velocity). Results are mean±s.d. of three experiments. *P<0.05, ***P<0.005 (Student's t-test). (C) Fold change (mean±s.d., n=2 separate experiments) of 6E10 colocalized with LAMP1.

Fig. 6.

Reduced Aβ40 secretion and increased intracellular APP and/or Aβ after depletion of Hrs or Tsg101. (A) ELISA shows reduced Aβ40 secretion in N2a-APP cells depleted of of Hrs (siHrs) or Tsg101 (siTsg101). (B) Hrs or Tsg101 depletion does not affect release of LDH activity into culture medium. (C–G) N2a-APP cells were depleted of Hrs or Tsg101 and media (C,D) and cell lysates (E–G) were analysed by western blotting. All results are mean±s.d. of three experiments. *P<0.05, ***P<0.005 (Student's t-test).