Analysis of novel endosome-to-Golgi retrieval genes reveals a role for PLD3 in regulating endosomal protein sorting and amyloid precursor protein processing
Analysis of novel endosome‑to‑Golgi retrieval genes reveals a role for PLD3 in regulating endosomal protein sorting and amyloid precursor protein processing
Aamir S. Mukadam 0
Sophia Y. Breusegem 0
Matthew N. J. Seaman 0
-phosphate receptor GFP Green fluorescent protein 0
Sortilin-like 0
0 Cambridge Institute for Medical Research, Cambridge Biomedical Campus, University of Cambridge , Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY , UK
The processing of amyloid precursor protein (APP) to the neurotoxic pro-aggregatory Aβ peptide is controlled by the mechanisms that govern the trafficking and localisation of APP. We hypothesised that genes involved in endosomal protein sorting could play an important role in regulating APP processing and, therefore, analysed ~ 40 novel endosome-to-Golgi retrieval genes previously identified in a genome-wide siRNA screen. We report that phospholipase D3 (PLD3), a type II membrane protein, functions in endosomal protein sorting and plays an important role in regulating APP processing. PLD3 co-localises with APP in endosomes and loss of PLD3 function results in reduced endosomal tubules, impaired trafficking of several membrane proteins and reduced association of sortilin-like 1 with APP.
Phospholipase D; Endosome; Amyloid precursor protein; SorL1; Alzheimer disease
Introduction
The processing of amyloid precursor protein (APP) to form
the neurotoxic pro-aggregatory Aβ peptide is believed to be
a key initiating event in the pathogenesis of Alzheimer’s
disease [
1
]. The trafficking and localisation of APP within the
post-Golgi endocytic system plays an important role in
regulating the exposure of APP to the secretases that mediate
its cleavage to form Aβ peptides [
2, 3
]. Most evidence now
supports a model whereby the cleavage of APP to generate
Aβ occurs in an endocytic compartment where β-secretase
(BACE) is predominately localised [4]. Thus, mechanisms
that direct APP away from endosomes towards either the
Golgi complex or the cell surface are considered to be
protective of APP processing to Aβ [
5, 6
].
The retromer complex is a key mediator of endosomal
protein sorting and has been shown to operate in both
endosome-to-Golgi and endosome-to-cell surface traffic,
regulating the localisation of membrane proteins such as the
cation-independent mannose 6-phosphate receptor (CIMPR),
sortilin and Glut-1 [
7
]. The retromer complex comprises a
stable trimeric protein complex containing Vps35, Vps26
and Vps29 that together select membrane proteins (‘cargo’)
for packaging into tubular carriers that form through the
action of the other functional unit of retromer, the sorting
nexin (Snx) dimer. For endosome-to-Golgi traffic, the Snx
dimer contains one copy of either Snx1 or Snx2 paired with
either Snx5 or Snx6 [
8, 9
].
Another notable cargo protein for retromer is the
membrane protein SorL1 (also known as SorLA) that traffics
from endosomes to the Golgi in a retromer-dependent
manner. SorL1 can directly associate with APP and can bind to
the retromer cargo-selective trimer through an interaction
with Vps26. SorL1 can thereby direct APP into the
retromermediated endosome-to-Golgi retrieval pathway thus
protecting APP from cleavage by BACE [
10–12
]. Mutations in
SorL1 can cause late-onset AD (LOAD) [
13, 14
] and
variants of genes that regulate recruitment of the retromer
complex to endosomes have been shown to predispose to LOAD
[15]. Furthermore, a point mutation in the retromer protein,
Vps35, that results in the protein becoming unstable, may
be causal in early-onset AD [
16
]. Also, it has been shown
that expression levels of retromer proteins are reduced in
the brains of AD patients and that loss of retromer function
results in increased processing of APP to Aβ [
17, 18
].
Due to its key function in endosomal protein sorting and
prominent role in regulating APP trafficking, there has been
considerable interest in retromer as a potential engine of
pathogenesis for AD [
19, 20
], but retromer does not operate
in isolation in endosomal protein sorting. We have recently
reported the results of a genome-wide siRNA screen for
novel endosome-to-Golgi retrieval genes that may function
alongside retromer [21]. Among the genes identified as new
players in the endosome-to-Golgi pathway were a surprising
number of multi-pass membrane-spanning proteins
including SFT2D2 and ZDHHC5. These two proteins are required
for the endosome-to-Golgi retrieval of the CIMPR and both
SFT2D2 and ZDHHC5 are localised to endosomes positive
for retromer proteins. We hypothesised that any of the genes
identified as novel endosome-to-Golgi retrieval genes may
encode proteins that could function in endosomal protein
sorting and may, therefore, regulate APP localisation and
processing. We have undertaken an analysis of the
endosome-to-Golgi retrieval genes and identified those genes
that, when silenced, result in increased processing of APP
to Aβ. We report that among the (...truncated)