Abstract
Alzheimer’s disease (AD) is characterized by the buildup of amyloid-β peptides (Aβ) aggregates derived from proteolytic processing of the β-amyloid precursor protein (APP). Amyloidogenic cleavage of APP by β-secretase/BACE1 generates the C-terminal fragment C99/CTFβ that can be subsequently cleaved by γ-secretase to produce Aβ. Growing evidence indicates that high levels of C99/CTFβ are determinant for AD. Although it has been postulated that γ-secretase-independent pathways must control C99/CTFβ levels, the contribution of organelles with degradative functions, such as the endoplasmic reticulum (ER) or lysosomes, is unclear. In this report, we investigated the turnover and amyloidogenic processing of C99/CTFβ in human H4 neuroglioma cells, and found that C99/CTFβ is localized at the Golgi apparatus in contrast to APP, which is mostly found in endosomes. Conditions that localized C99/CTFβ to the ER resulted in its degradation in a proteasome-dependent manner that first required polyubiquitination, consistent with an active role of the ER associated degradation (ERAD) in this process. Furthermore, when proteasomal activity was inhibited C99/CTFβ was degraded in a chloroquine (CQ)-sensitive compartment, implicating lysosomes as alternative sites for its degradation. Our results highlight a crosstalk between degradation pathways within the ER and lysosomes to avoid protein accumulation and toxicity.
Highlights
Alzheimer’s disease (AD) is characterized by the accumulation of aggregated amyloid-b (Ab) peptide species derived from successive proteolytic cleavages of the b-amyloid precursor protein (APP) [1]
In cells treated with DAPT, C99-GFP had a longer half-life of,54 min, AICDcGFP was almost absent, and there was a steady accumulation of C83-GFP (Fig. 1, C and D, DAPT), which is the product of the non-amyloidogenic proteolytic processing of C99-GFP by asecretase [18]
This is mainly because several studies have shown C99 generation at different intracellular sites [19,36,38,39,40,41,42], and other studies have reported different subcellular distribution of the secretases [2,5,17,22,42]. The reason for this debate lies in part in findings showing that Brefeldin A (BFA) greatly reduces the levels of C99 and Ab, leading to the notion that APP is not cleaved by b-secretase within the endoplasmic reticulum (ER) [30,43]
Summary
Alzheimer’s disease (AD) is characterized by the accumulation of aggregated amyloid-b (Ab) peptide species derived from successive proteolytic cleavages of the b-amyloid precursor protein (APP) [1]. Several reports have postulated that C99 levels are regulated by c-secretase-independent pathways [4,5,6,7,8,9]; the contribution of these degradation pathways, such as those working in the endoplasmic reticulum (ER) or in lysosomes, to the turnover of C99 and Ab production is still unclear. Accumulation of APP at the ER results in the production of the N-terminal soluble fragment generated by b-secretase [15]. We found that C99 is actively degraded at the ER in an ubiquitin and proteasome dependent manner, requiring polyubiquitination of its cytosolic lysine residues. We observed that degradation of C99 within acidic compartments, in response to proteasome inhibition, was not dependent on its cytosolic lysine residues, indicating that C99 is degraded in lysosomes in a ubiquitin-independent manner. We found that delivery of C99 to the plasma membrane was diminished in the absence of cytosolic lysine residues, instead resulting in a strong accumulation of C99 at the
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