Abstract
Amyloid precursor protein (APP) is processed along the amyloidogenic pathway by the β-secretase, BACE1, generating β-amyloid (Aβ), or along the nonamyloidogenic pathway by α-secretase, precluding Aβ production. The plasma membrane is considered the major site for α-secretase-mediated APP cleavage, but other cellular locations have not been rigorously investigated. Here, we report that APP is processed by endogenous α-secretase at the trans-Golgi network (TGN) of both transfected HeLa cells and mouse primary neurons. We have previously shown the adaptor protein complex, AP-4, and small G protein ADP-ribosylation factor-like GTPase 5b (Arl5b) are required for efficient post-Golgi transport of APP to endosomes. We found here that AP-4 or Arl5b depletion results in Golgi accumulation of APP and increased secretion of the soluble α-secretase cleavage product sAPPα. Moreover, inhibition of γ-secretase following APP accumulation in the TGN increases the levels of the membrane-bound C-terminal fragments of APP from both α-secretase cleavage (α-CTF, named C83 according to its band size) and BACE1 cleavage (β-CTF/C99). The level of C83 was ∼4 times higher than that of C99, indicating that α-secretase processing is the major pathway and that BACE1 processing is the minor pathway in the TGN. AP-4 silencing in mouse primary neurons also resulted in the accumulation of endogenous APP in the TGN and enhanced α-secretase processing. These findings identify the TGN as a major site for α-secretase processing in HeLa cells and primary neurons and indicate that both APP processing pathways can occur within the TGN compartment along the secretory pathway.
Highlights
Amyloid precursor protein (APP) is processed along the amyloidogenic pathway by the -secretase, BACE1, generating -amyloid (A), or along the nonamyloidogenic pathway by ␣-secretase, precluding A production
Our findings demonstrate that ␣-secretase cleavage of APP occurs in the trans-Golgi network (TGN); the TGN may represent a major site for nonamyloidogenic processing
1) Depletion of AP-4 or ADP-ribosylation factor–like GTPase 5b (Arl5b) retarded the exit of APP from the TGN, resulting in APP accumulation in the TGN, and dramatically enhanced the cleavage of APP by endogenous ␣-secretase, as well as BACE1, in stable HeLa cells expressing APP695WT and primary neurons
Summary
The trans-Golgi network is a major site for ␣-secretase processing of amyloid precursor protein in primary neurons. ADAM10 is synthesized as an inactive preprotein and is activated following cleavage by a convertase within the secretory pathway, most likely in the late Golgi and/or during transport from the Golgi to the cell surface [19, 20] It is not known whether ␣-secretase cleavage of APP can occur at the TGN or is restricted to post-Golgi compartments. Recent studies have demonstrated that newly synthesized APP is diverted at the TGN and trafficked directly to early endosomes, with only low levels detected at the cell surface [11] Given these issues, the potential importance of intracellular sites of ␣-secretase cleavage of APP remains an open question and warrants further investigation. We have shown that retarding the exit of APP from the Golgi resulted in enhanced processing of APP by ␣-secretase in both transfected HeLa cells and primary mouse cortical neurons demonstrating that both amyloidogenic and nonamyloidogenic APP-processing pathways can occur in the late Golgi
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