Abstract
Amyloid plaques, a neuropathological hallmark of Alzheimer's disease, are largely composed of amyloid β (Aβ) peptide, derived from cleavage of amyloid precursor protein (APP) by β- and γ-secretases. The endosome is increasingly recognized as an important crossroad for APP and these secretases, with major implications for APP processing and amyloidogenesis. Among various post-translational modifications affecting APP accumulation, ubiquitination of cytodomain lysines may represent a key signal controlling APP endosomal sorting. Here, we show that substitution of APP C-terminal lysines with arginine disrupts APP ubiquitination and that an increase in the number of substituted lysines tends to increase APP metabolism. An APP mutant lacking all C-terminal lysines underwent the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aβ40. Artificial APP ubiquitination with rapalog-mediated proximity inducers reduced Aβ40 generation. A lack of APP C-terminal lysines caused APP redistribution from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with a subsequent decrease in APP C-terminal fragment (CTF) content in secreted exosomes, but had minimal effects on APP lysosomal degradation. Both the increases in secreted and intracellular Aβ40 were abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared with PSEN1. Our findings demonstrate that ubiquitin can act as a signal at five cytodomain-located lysines for endosomal sorting of APP. They further suggest that disruption of APP endosomal sorting reduces its sequestration in ILVs and results in PSEN2-mediated processing of a larger pool of APP-CTF on the endosomal membrane.
Highlights
Amyloid plaques, a neuropathological hallmark of Alzheimer’s disease, are largely composed of amyloid  (A) peptide, derived from cleavage of amyloid precursor protein (APP) by and ␥-secretases
APP constructs with individual mutations K724R, K725R, and K726R allow us to identify whether all three of these residues are necessary for APP ubiquitination
We show that the ubiquitin signal in the anti-GFP immunoprecipitate of HEK293T cells co-transfected with APPGFP and UbHA is a smear above ϳ130 kDa, suggesting multi- or polyubiquitination of APPWTGFP (Fig. 1B)
Summary
To assess whether the five lysines present in the APP C-terminal domain (Fig. 1A) are sites of ubiquitination, we generated a panel of GFP-tagged APP mutants with each lysine mutated to arginine individually and in several combinations. To determine whether the disrupted ILV sorting of APP5RGFP leads to a deficiency of APP on exosomes, we expressed the APPGFP mutants in N2a cells and collected and purified exosomes from the cell culture media (Fig. 8A and as characterized in Ref. 54). No significant changes were observed for A42 These results corroborate our hypothesis that the higher A40 levels measured in APP5R-expressing cells are due to increased cleavage by PSEN2-containing ␥-secretase complexes. Sannerud et al [43] showed that PSEN1 contributes more to extracellular A secretion than PSEN2, our data suggest that PSEN2-containing ␥-secretase complexes on the endosomal limiting membrane cleave an additional pool of APP-CTFGFP that accumulates when APP5RGFP fails to be sequestered in ILVs. Unexpectedly, the reduction in the APP5RGFP A42/ A40 ratio was unchanged upon PSEN2 depletion, there was a greater and more significant reduction in the APPWTGFP condition. Our data suggest that the specific increase in A40 exhibited by APP5RGFP is primarily due to cleavage of APP5RGFP by PSEN2-containing ␥-secretase complexes subsequent to endosomal redistribution of the ubiquitin-deficient APP mutant
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