From the Fisher Center for Research on Alzheimer's
Disease and Laboratory of Molecular and Cellular Neuroscience, The
Rockefeller University, New York, New York 10021, the
§ Department of Neurobiology, Pharmacology and Physiology,
the University of Chicago, Chicago, Illinois 60637, and
Huffington Center on Aging, Department of Molecular and Human
Genetics, Baylor College of Medicine, Houston, Texas 77030
Received for publication, September 5, 2002, and in revised form, October 31, 2002
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ABSTRACT |
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Presenilins (PS1/PS2) play a critical role in
proteolysis of Alzheimer's disease
(AD)1 is characterized by the
excessive generation and accumulation of Expression of autosomal dominant variants of either Although it has generally been accepted that PSs are essential for
Based on these observations, we investigated the potential role of PS1
in regulating intracellular trafficking of full-length Cell Lines--
Mouse N2a neuroblastoma cells doubly transfected
with cDNAs encoding human Neuronal Culture--
Human wt PS1 (line 17-3) or FAD-linked PS1
A246E mutant transgenic mice (line 16-4) (34) were mated with
heterozygous PS1 null mice (PS1+/ Preparation of Permeabilized N2a Cells--
It has been well
established that incubation of cells at 15 (36) or 20 °C (30) leads
to an accumulation of membrane and secretory proteins in the ER and
TGN, respectively. To assay Formation of Nascent Secretory Vesicles in Permeabilized Cells
and Immunoprecipitation--
Following incubation of cell-free
systems, vesicle and membrane fractions were separated by
centrifugation at 11,000 rpm for 30 s at 4 °C in a Brinkmann
centrifuge (Brinkmann Instruments). Vesicle (supernatant) and membrane
(pellet) fractions were diluted with IP buffer (50 mM
Tris-HCl, pH 8.8, 150 mM NaCl, 6 mM EDTA, 2.5%
Triton X-100, 5 mM methionine and cysteine, and 1 mg/ml
bovine serum albumin), immunoprecipitated using Biotinylation and Detection of Cell Surface Immunofluorescence Confocal Microscopy--
For staining of
full-length PS1 Deficiency Leads to Increased
The formation of
We next tested whether PS1 deficiency might affect
To support further the notion that Loss-of-Function PS1 Mutants Accelerate
Together, these results suggest that the loss of function for PS1 in
Loss-of-Function PS1 Mutations Increase the Amount of Full-length
FAD-linked PS1 Mutants Delay
We further tested whether FAD-linked PS1 mutations affect
To ascertain the selectivity of impaired FAD-linked PS1 Mutants Lower the Amount of Full-length
To examine directly the delivery of newly synthesized full-length
An FAD-linked PS1 Variant Causes a Profound Reduction of Cell
Surface-bound
Embryonic cortical neurons from PS1 knockout mouse embryos rescued with
comparable levels of expression of either wt human PS1 (line 17-3) or
FAD-linked PS1 A246E mutant (line 16-4) (34) were grown in culture, and
To date, the mechanisms by which PSs exert their effects on The above findings are consistent with a model in which PS1 might
regulate -amyloid precursor protein (
APP) to generate
-amyloid, a peptide important in the pathogenesis of Alzheimer's
disease. Nevertheless, several regulatory functions of PS1 have also
been reported. Here we demonstrate, in neuroblastoma cells, that PS1
regulates the biogenesis of
APP-containing vesicles from the
trans-Golgi network and the endoplasmic reticulum. PS1
deficiency or the expression of loss-of-function variants leads to
robust vesicle formation, concomitant with increased maturation and/or
cell surface accumulation of
APP. In contrast, release of vesicles
containing
APP is impaired in familial Alzheimer's disease
(FAD)-linked PS1 mutant cells, resulting in reduced
APP delivery to
the cell surface. Moreover, diminution of surface
APP is profound at
axonal terminals in neurons expressing a PS1 FAD variant. These results
suggest that PS1 regulation of
APP trafficking may represent an
alternative mechanism by which FAD-linked PS1 variants modulate
APP processing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid peptides (A
).
The amyloidogenic A
peptide is proteolytically derived from the
-amyloid precursor protein (
APP) within the secretory pathway by
distinct enzymatic activities known as
- and
-secretase (1, 2).
Full-length
APP is synthesized in the endoplasmic reticulum (ER) and
transported through the Golgi apparatus. The major population of
secreted A
peptides is generated within the trans-Golgi
network (TGN) (3-5), also the major site of
APP residence in
neurons at steady state.
APP can be transported in TGN-derived
secretory vesicles to the cell surface if not first proteolyzed to A
or an intermediate metabolite. At the plasma membrane
APP is either
cleaved to produce a soluble molecule, s
APP (6) or, alternatively,
reinternalized within clathrin-coated vesicles to an
endosomal/lysosomal degradation pathway (7, 8). Thus, the distribution
of
APP between the TGN and cell surface has a direct influence upon
the relative generation of s
APP versus A
. This
phenomenon makes delineation of the mechanisms responsible for
regulating
APP trafficking from the TGN relevant to understanding
the pathogenesis of AD.
APP, presenilin
1 (PS1), or presenilin 2 (PS2) results in increased A
42 production
and predispose individuals to early onset familial Alzheimer's disease
(FAD) (9-11). Presenilins (PSs), multitransmembrane proteins,
accumulate as endoproteolyzed heterodimers of N- and C-terminal
fragments and associate with other membrane proteins (e.g.
nicastrin, APH-1, and PEN-2) to form high molecular weight complexes
(9, 12-16). Several lines of evidence suggest that presenilin
complexes play a crucial role in intramembranous
-secretase cleavage
of type I membrane proteins including
APP and the signaling receptor, Notch-1. For example, genetic ablation of PS1, PS2, or other
components of the PS complex dramatically impairs A
secretion and
production of the Notch derivative, S3/NICD, in cells (14, 15, 17, 18).
Mutations of conserved transmembrane aspartate residues in PSs result
in loss-of-function leading to reduced A
secretion (19, 20).
Finally, biochemical fractionation studies closely link PSs with
-secretase activity, and
-secretase inhibitors can be
photo-cross-linked to PS1 and PS2 (21, 22).
-cleavage, it has not been firmly established that PSs are the
catalytic component of the enzyme complex. For example, recent studies
(23) have shown that the production of A
42 in early compartments of
the secretory apparatus is unimpaired in the absence of PSs. Whereas
the hypothesis remains attractive that PSs are the
-secretase,
several reports indicate that PSs mediate additional physiological
functions, including roles in calcium homeostasis, neurite outgrowth,
apoptosis, and synaptic plasticity (24), and some of these functions
are influenced by FAD-linked PS mutations. PS1 has been implicated in
regulating intracellular trafficking and maturation of selected
transmembrane proteins. PS1 deficiency significantly affects
trafficking of the tyrosine kinase receptor TrkB, as well as the
PS1-interacting protein ICAM-5/telecephalin (25, 26). Indeed evidence
has emerged to support the notion that PS1 may facilitate
-secretase cleavage of substrates via regulating the maturation and intracellular trafficking of substrates and/or components of the
-secretase complex. For example, expression of the PS1 aspartate variants leads to
accumulation of
APP C-terminal fragments (CTFs) as well as
full-length
APP at the cell surface (20, 27). Several recent studies
(28, 29) further demonstrate that PS1 regulates the maturation and cell
surface accumulation/trafficking of nicastrin.
APP through
the secretory pathway. We previously established a cell-free system in
which we can reconstitute both the formation of A
and the
trafficking of
APP/A
from the ER or the TGN (3, 5, 30-32). By
utilizing this system, we directly examined individual cellular
processes involved in PS1 regulation of
APP trafficking. We report
here that PS1 deficiency or expression of loss-of-function variants led
to robust formation of
APP-containing vesicles, concomitant with
increased maturation and/or cell surface accumulation of
APP. In
contrast, vesicle formation from the TGN and the ER was impaired in
cells expressing FAD-linked PS1 mutants, resulting in a reduction of
APP delivery to the cell surface. We also observed a profound
reduction of surface
APP at axonal terminals in neurons harboring an
FAD PS1 mutation (A246E), compared with neurons expressing wild type
(wt) PS1. Taken together, these results suggest that PS1 can modulate
metabolism of
APP via regulating
APP trafficking within the
secretory pathway and thus affect A
generation by controlling the
availability of substrate
APP to appropriate secretases.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
APP harboring the "Swedish" double
mutant (
APPswe) and human wt PS1 or various PS1 mutants (11, 30)
were maintained in medium containing 50% Dulbecco's modified Eagle's
medium, 50% Opti-MEM, supplemented with 5% fetal bovine serum,
antibiotics, and 200 mg/ml G418 (Invitrogen). Immortalized
PS1
/
fibroblasts (33) were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and antibiotics. To circumvent potential variability
between fibroblasts established from PS1+/+ and
PS1
/
embryos, immortalized
PS1
/
fibroblasts were transfected with wt
PS1 expression plasmid, and stable transfectants were isolated in 400 mg/ml Zeocin (Invitrogen). To express
APPswe,
PS1
/
fibroblasts and wt PS1 transfectants
were infected with
APPswe retroviruses produced from the mouse
NIH3T3-based PT67 packaging cell line, and stable expressors
(designated PS1
/
and wt) were isolated in
media containing 2 mg/ml puromycin (Calbiochem), and ~150-200
colonies were pooled for further studies. Blastocyst-derived wt and
PS1
/
/PS2
/
cells
have been described previously (28).
) (35).
Double heterozygous F1s were then intercrossed to generate offspring
that express human wt or A246E PS1 in the
PS1
/
background. Genotypes were determined
by PCR amplification as described previously (34). Primary neuronal
cultures were derived from the cerebral cortices of embryonic day 17 (E17) mouse embryos from the above matings. Dissociated neurons were
plated (~5 × 104 cells per chamber) on
poly-L-lysine (Sigma)-precoated 8-chamber slides (Lab-Tek)
in serum-free neurobasal medium with N2 supplement as well
as B27 supplement (Invitrogen) and cultured for 7 days. Media were
replaced every 2 days with the addition of 16.5 mg/ml uridine and 6.7 mg/ml 5-fluoro-2'-deoxyuridine to prevent proliferation of glial cells.
APP trafficking from TGN, cells were
pulse-labeled with [35S]methionine (500 µCi/ml) for 15 min at 37 °C, washed with phosphate-buffered saline (prewarmed to
20 °C), and chased for 2 h at 20 °C in prewarmed complete
media. To assay
APP trafficking from the ER, cells were pulse-labeled for 4 h at 15 °C. For both types of preparations, cells were permeabilized at the termination of incubation as follows. Cells were first incubated at 4 °C in "swelling buffer" (10 mM KCl, 10 mM HEPES, pH 7.2) for 10 min. The
buffer was discarded and replaced with 1 ml of "breaking
buffer" (90 mM KCl, 10 mM HEPES, pH 7.2),
after which the cells were broken by scraping with a rubber policeman.
Cells were centrifuged at 800 × g for 5 min, washed in
5 ml of breaking buffer, and resuspended in 5 volumes of breaking
buffer. This results in >95% cell breakage as evaluated by trypan
blue staining. Broken cells (cell-free system) were incubated in a
final volume of 300 µl containing 2.5 mM
MgCl2, 0.5 mM CaCl2, 110 mM KCl, cytosol (30 µg protein) prepared from N2a cells
(32, 37), and an energy-regenerating system consisting of 1 mM ATP, 0.02 mM GTP, 10 mM creatine
phosphate, 80 µg/ml creatine phosphokinase, and a protease inhibitor
mixture. Incubations were carried out at 37 °C for various times
(15-120 min) to observe the kinetics of protein trafficking.
APP C-terminal
antibody 369 (7, 38) or anti-NCAM and anti-FGFR1 antibodies (Santa Cruz
Biotechnology), and analyzed by SDS-PAGE. Each experiment was performed
at least three times. Band intensities were analyzed and quantified
using NIH ImageQuant software, version 1.52.
APP--
Stably
transfected N2a cells were labeled with [35S]methionine
(500 µCi/ml) for 10 min at 37 °C and chased for 2 h at
20 °C in complete medium to accumulate labeled proteins in the TGN. To restore vesicle trafficking from TGN, cells were transferred to
37 °C for various time intervals. Cells were then incubated at
4 °C with 0.5 mg ml
1
sulfo-N-hydroxysuccinimide biotin (Pierce) to biotinylate
cell surface proteins. Biotinylated and non-biotinylated proteins were first separated into two fractions by binding to streptavidin-agarose beads (Pierce), and
APP from each fraction was immunoprecipitated with antibody 369 (7, 38) and analyzed by SDS-PAGE (39).
APP, N2a cells grown in chamber slides were incubated at
4 °C with primary antibody 6E10 (diluted 1:100 in growth medium,
Senetek PLC) for 1 h without fixation or permeabilization.
Following incubation with secondary antibodies and FITC-conjugated
Vicia villosa agglutinin (1:100, Vector Laboratories Inc.),
cells were fixed with 4% formaldehyde at room temperature for 15 min.
Cultured neurons were fixed twice with 4% formaldehyde for 15 min
twice. For surface staining of full-length
APP, cells were directly
incubated with primary antibody mAb348 (1:100 dilution, Roche Molecular
Biochemicals) at 4 °C overnight. In some experiments, neurons were
permeabilized by ice-cold methanol for 2 min prior to overnight
incubation with mAb348 or anti-GAP43 antibody (1:4000). Immunofluorescence staining was examined by confocal microscopy (LSM510, Zeiss).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
APP Transport from TGN to
Plasma Membrane and from ER to Golgi--
It has been well established
that PS1-deficient neurons fail to secrete A
but accumulate
intracellular
APP C-terminal fragments (25, 40). In addition to
affecting
-secretase activity, recent observations (25, 28, 29) that
PS1 deficiency or loss-of-function alters the maturation and cell
surface accumulation of certain membrane proteins, such as
APP,
nicastrin, and Notch-1, suggest a potential role for PS1 in
intracellular protein trafficking. To support further the notion that
wt PS1 may have a direct regulatory effect on
APP trafficking
through secretory compartments, we assessed the formation of
APP-containing vesicles from the TGN and from the ER in
PS1
/
fibroblasts using a cell-free
reconstitution system.
APP-containing vesicles from the TGN was examined
using a cell-free system that has been used extensively to study
APP
trafficking and A
generation (see "Experimental Procedures" and
Refs. 3, 5, and 32). This cell-free system has been used to investigate
nascent secretory vesicle budding from a variety of cells (30, 31), and
the integrity of TGN stacks and derived vesicles has been demonstrated
by electron microscopy (37). To study trafficking of
APP-containing
vesicles from the TGN, we labeled wt and
PS1
/
/PS2
/
fibroblasts with [35S]methionine and then incubated the
cells at 20 °C, a temperature at which transport of proteins,
including
APP, from the ER to the TGN is unimpaired. However, under
these conditions, the egress of secretory vesicles from the TGN is
blocked, thus allowing labeled
APP (and other membrane proteins) to
accumulate in the TGN (3, 5, 32). This experimental design ensures that
TGN-specific vesicle biogenesis is measured. In our cell-free
trafficking assays, following permeabilization and incubation at
37 °C, budding of
APP-containing vesicles from the TGN was
greatly increased at all time points examined in preparations that
lacked PS1 when compared with preparations from cells that express wt
PS1 (Fig. 1, a and
b). After 2 h of incubation, the amount of
APP
transported out of the TGN was 54.2% higher in
PS1
/
cell preparations compared with
preparations from PS1 wt cells (i.e. 21.9 versus
14.2% of the total labeled
APP in the TGN).
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Fig. 1.
PS1 deficiency accelerates
APP transport from the TGN and from the ER.
a-d, PS1
/
fibroblasts expressing
human
APPswe alone (PS1
/
) or coexpressing
APPswe and wt human PS1 (PS1WT) were labeled for 15 min
with [35S]methionine at 37 °C and chased for 2 h
at 20 °C to accumulate labeled
APP in the TGN. Alternatively,
cells were labeled for 4 h at 15 °C to accumulate labeled
APP within the ER. Permeabilized cells were prepared and incubated
at 37 °C for various times to allow the formation of post-TGN
(a and b) or post-ER vesicles (c and
d). Labeled
APP was immunoprecipitated from nascent
vesicles or the donor compartments and analyzed by SDS-PAGE and
autoradiography (a and c). The kinetics of
APP-containing vesicle formation is presented as percent of vesicle
budding (b and d); data represent mean ± S.E. from three independent experiments. e, the kinetics of
ER budding of FGFR1-containing vesicles was analyzed as
above. Data represent mean ± S.E. of three experiments.
f, samples of cell lysates with equal amount of protein (50 µg protein/lane) prepared from wt and
PS1
/
/PS2
/
cells
were analyzed by Western blotting using C-terminal
APP antibody
CT15.
APP vesicle
transport from the ER to Golgi utilizing a modified cell-free system in
which
APP-containing post-ER vesicles were reconstituted. In this
case, cells were labeled with [35S]methionine at 15 °C
to accumulate
APP within the ER (see "Experimental Procedures"
and Ref. 5), followed by permeabilization and incubation at 37 °C to
initiate vesicle release. The budding of
APP-containing ER vesicles
was accelerated (~2-fold) in PS1
/
cells
compared with wt cells (Fig. 1, c and d). To
assess whether PS1 deficiency selectively affects
APP trafficking,
budding of fibroblast growth factor receptor
(FGFR1)-containing vesicles from the ER was determined in
parallel and remained unchanged by PS1 deficiency (Fig. 1e).
Collectively these results suggest that PS1 selectively regulates
APP trafficking from ER and TGN compartments.
APP is transported more
efficiently out of the ER and Golgi compartments in the absence of PS,
we examined the patterns of
APP maturation in detergent lysates
prepared from
PS1
/
/PS2
/
cells
and wt fibroblasts. As expected from the
PS1
/
-permeabilized cell studies, the amount
of mature (glycosylated/sialylated)
APP in
PS1
/
/PS2
/
cells
was significantly higher than that in PS1 wt cells (Fig. 1f), indicative of increased residence and transit of
APP
through the late secretory compartments. However, alternation in
APP glycosylation/maturation was much less dramatic in
PS1
/
cells than in
PS1
/
/PS2
/
cells
(data not shown). Although the underlying mechanism is not clear, this
may be attributed to differential roles of PS1 and PS2 in APP
trafficking versus maturation.
APP Trafficking from the
ER without Altering TGN Vesicle Budding--
To confirm further that
loss of PS1 activities results in accelerated
APP trafficking
through the secretory pathway, N2a cells expressing loss-of-function
PS1 variants were examined to assess their effects on intracellular
transport of
APP. Previous studies demonstrated that a single amino
acid substitution in PS1 transmembrane domain 7 (D385A) (19, 20) or
deletion of the first two PS1 transmembranes (
1,2) (41) lowers A
secretion and leads to accumulation of
APP
CTFs. Unexpectedly,
APP-containing vesicle budding from the TGN was not changed by the
expression of PS1 loss of function mutations (D385A or
1,2) (~35%
of maximal
APP vesicle budding in both D385A and
1,2
versus ~38% in PS1 wt cells) (Fig.
2, a and b).
However, the trafficking of
APP from the ER to Golgi was
significantly increased in these loss-of-function PS1 mutants (Fig. 2,
c and d). The maximal level of vesicle budding was increased by ~1.5- (for D385A) to 2-fold (for
1,2) when
compared with PS1 wt; this increase in budding is similar to that
observed in PS1-deficient cells. As a result of accelerated ER
trafficking, the total amount of
APP residing in TGN membrane in
loss-of-function PS1 mutants was increased compared with that in wt TGN
membrane (Fig. 2a), although the rate of budding of
APP-containing vesicles from the TGN was unchanged (Fig.
2b).
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Fig. 2.
APP trafficking from the ER and
the TGN in cells expressing loss-of-function mutations of PS1.
Stable N2a cells coexpressing
APPswe and wt PS1 (WT) or
PS1 harboring either D385A or
1,2 mutations were used. Budding
assays were performed as described in Fig. 1. Quantitative data
represent mean ± S.E. from three independent experiments.
-cleavage of
APP and the complete absence of PS1 protein differentially regulate vesicle biogenesis from the TGN, although the
efflux of
APP molecules from the ER is enhanced under both conditions.
APP Delivered to the Plasma Membrane--
Despite the lack of
marked differences in TGN vesicle biogenesis, increased ER to Golgi
trafficking of
APP observed in the loss-of-function mutant cells may
be sufficient to elevate the steady-state levels of
APP at the cell
surface in intact cells. In addition, as recently reported (42), a
delay in the internalization of surface-bound
APP may further
facilitate increased surface accumulation of
APP. Indeed, live
staining of
1,2 cells using monoclonal antibody 6E10 revealed an
obvious increase in the amount of surface-bound
APP compared with
PS1 wt cells. The amount of total surface glycoproteins is identical in
the two types of cells as judged by staining for V. villosa
agglutinin, a lectin that binds to
N-acetyl-D-galactosamine linked to serine or
threonine residues in glycoproteins (43) (Fig.
3a). The amount of newly synthesized
APP delivered to the cell surface was measured
quantitatively in these cells by pulse-chase labeling in combination
with cell-surface biotinylation (39) in intact cells. Cells were
pulse-labeled for 10 min with [35S]methionine at 37 °C
and chased for 2 h at 20 °C to accumulate labeled
APP in the
TGN. Subsequent incubation at 37 °C allows trafficking of
APP
from TGN to cell surface in a synchronized fashion (39). Newly arrived
[35S]methionine-labeled cell surface
APP molecules
were then labeled with biotin at 4 °C for 15 min and separated from
TGN-associated
APP by streptavidin bead precipitation. Up to 14.3%
of nascent
APP was transported to the plasma membrane after 120 min
of chase, a value that is 48.9% greater than that in PS1 wt cells
(Fig. 3, b and c). The findings from
loss-of-function PS1 mutants together with studies using PS1-deficient
cells strongly suggest that PS1 plays a role in regulating the delivery
of nascent
APP molecules to the cell surface.
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Fig. 3.
Delivery of full-length
APP to the plasma membrane in cells harboring
loss-of-function PS1 mutations. a, live N2a cells were
incubated with primary antibody 6E10 (1:100) at 4 °C for 1 h to
label cell surface
APP (red) and FITC-conjugated V. villosa agglutinin to stain all surface glycoproteins
(green). Cells were then fixed and visualized by confocal
microscopy. b and c, cells were labeled with
[35S]methionine at 37 °C for 10 min and chased at
20 °C for 2 h to accumulate labeled
APP in the TGN (total
cell
APP). Cells were then incubated at 37 °C for various times
to allow transport of
APP to the plasma membrane. Cell surface
proteins were then biotinylated at 4 °C for 15 min. Biotinylated and
non-biotinylated proteins were separated into two fractions by binding
to streptavidin beads.
APP was immunoprecipitated from each fraction
and analyzed. Quantitative data represent mean ± S.E. from three
independent experiments.
APP Transport from the TGN and the
ER--
We next investigated whether FAD-linked PS1 variants might
influence
APP trafficking. The kinetics of
APP-containing vesicle budding from the TGN of N2a cells expressing wt PS1 was compared with
that of cells expressing FAD-linked PS1 variants. The budding of
TGN-derived
APP vesicles was dramatically impaired in FAD-linked PS1
mutants (
E9, A246E, and M146L) at all time points examined, compared
with PS1 wt cells. Among the three FAD-linked PS1 mutations examined,
PS1 A246E demonstrated the highest degree of impairment in
APP
vesicle budding from the TGN, with maximal
APP vesicle budding of
21.8% at 90 min compared with 38% in PS1 wt cells (Fig. 4, a and b).
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Fig. 4.
FAD-linked PS1 mutants delay APP transport
from the TGN and the ER. a-d, stable N2a cells coexpressing
APPswe and either wt PS1 (WT) or PS1 harboring one of the
indicated FAD-linked mutations were used in these experiments. Vesicle
budding experiments were performed as described in the legend to Fig.
1. Quantitative data represent mean ± S.E. from three independent
experiments. e, budding of NCAM-containing vesicles was
examined in parallel from WT and mutant cells and quantified.
APP
vesicle transport from the ER to Golgi. Similar to the results obtained
for TGN budding,
APP trafficking from the ER to Golgi, as judged by
the release of
APP-containing vesicles from the ER, was
significantly reduced in FAD-linked mutants, with a 50.9 and 57.9%
decrease in the maximal levels of vesicle budding in PS1
E9 and
A246E cells, respectively, compared with PS1 wt cells. The impairment
in ER vesicle biogenesis was relatively mild in the PS1 M146L mutation,
with an ~30% reduction compared with PS wt (Fig. 4, c and
d).
APP-containing vesicle
budding in cells expressing FAD-linked PS1 variants, we examined the
trafficking of neural cell adhesion molecule (NCAM) from the TGN using
the permeabilized cell system. In contrast to
APP, the rate of TGN
budding of vesicles containing NCAM isoforms was almost identical in
PS1 wt and mutant cells (Fig. 4e), suggesting that
FAD-linked PS1 variants specifically delay
APP transport from the
TGN to the cell surface.
APP
Delivered to the Plasma Membrane--
We reasoned that the alteration
in
APP trafficking by PS1 mutations seen in the permeabilized cell
system should affect the steady-state level of
APP on the plasma
membrane. To test this hypothesis we stained live N2a cells with
monoclonal antibody 6E10 to visualize surface-bound
APP and
APP
C-terminal fragments and FITC-conjugated V. villosa
agglutinin to label all glycoproteins. As shown in Fig.
5a, the intensity of
APP
immunofluorescence on the cell surface of mutant cells was markedly
reduced as compared with PS1 wt cells. V. villosa agglutinin
staining of surface glycoproteins was comparable between PS1 wt and
mutant cells (Fig. 5a). These findings are consistent with
the results from permeabilized cell experiments described above and
suggest that FAD-linked PS1 mutants impair the trafficking of
APP to
the cell surface.
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Fig. 5.
FAD-linked PS1 mutations reduce the amount of
full-length APP delivered to the plasma
membrane. Surface staining and budding assays were performed as
described in Fig. 3, except that N2a cells coexpressing
APPswe and
PS1 harboring one of the indicated FAD-linked mutations were used.
These assays were done in parallel to the experiments described in Fig.
3, and the WT data are the same as in Fig. 3. Quantitative data
represent mean ± S.E. from three independent experiments.
APP trafficking from the TGN to the plasma membrane, we carried out
pulse-chase labeling in combination with cell-surface biotinylation. As
shown in Fig. 5, b and c, in PS1 wt cells, 9.6% of nascent
APP that accumulated in the TGN during incubation at
20 °C left the TGN and traveled to the cell surface after a 2-h
chase at 37 °C. However, the amount of newly synthesized
APP that
accumulated on the cell surface at various chase time periods was much
lower in PS1 (
E9) mutant cells, with only 3-5% of nascent
APP
molecules delivered to the plasma membrane even after 2 h of
chase. Therefore, both immunofluorescence and biochemical studies indicate that the levels of full-length
APP delivered from TGN to
plasma membrane are diminished in cells expressing FAD-linked PS1
variants, most likely by delaying the budding of
APP-containing vesicles from the TGN and the ER.
APP at Axons and Axonal Terminals in Primary
Neurons--
Previous studies (44-48) have demonstrated that
APP
is axonally transported by the kinesin-mediated, fast anterograde
component. It has also been reported (49) that both full-length and
processed derivatives of
APP accumulate at presynaptic terminals of
cortical neurons. We further assessed whether FAD-linked mutations
affect the distribution of full-length
APP on the surface of primary neurons.
APP molecules on the cell surface at the axonal terminals and in the
cell body were visualized by immunofluorescence staining using
monoclonal antibody mAb348, which recognizes the ectodomain of
APP.
As shown in Fig. 6a, immunofluorescence of full-length
APP on the surface of live (non-permeabilized) wt PS1 rescued neurons was evident both in the cell
body and at the axonal terminals, whereas the immunofluorescence of
surface-bound
APP in PS1 A246E mutant rescued neurons was limited to
the cell body, being absent from axons and axonal terminals (Fig.
6a, top panels). As a control, live cells were
also doubly stained with V. villosa agglutinin to confirm
that the staining of surface-bound glycoproteins was comparable in wt
and mutant PS1 neurons (Fig. 6a, bottom panels).
However, after permeabilization, the distribution pattern of
intracellular full-length
APP was identical in PS1 wt and A246E
neurons (Fig. 6b, top panels). In addition,
intracellular localization of growth-associated protein 43 (GAP43), a
protein associated with growth cone membranes, was similar in wt and
mutant PS1 neurons (Fig. 6b, bottom panels). These data suggest that the FAD-linked mutants selectively impair the
targeting or fusion of
APP-containing vesicles to the plasma membrane, especially at axons and axonal terminals of neurons.
View larger version (39K):
[in a new window]
Fig. 6.
FAD-linked PS1 mutations cause more profound
reduction of surface-bound APP in axons and
axonal terminals. Primary neurons that express comparable levels
of either wt human PS1 or FAD-linked PS1 A246E mutant in
PS1
/
background were cultured for 7 days.
a, after 15 min of 4% formaldehyde fixation,
non-permeabilized cells were incubated with APP N-terminal antibody
mAb348 and FITC-conjugated V. villosa agglutinin.
b, in some experiments neurons were permeabilized with
ice-cold methanol for 2 min and then incubated with mAb348
(red) and anti-GAP43 (green) antibodies.
Immunofluorescence staining was visualized by confocal
microscopy.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
APP
metabolism are not fully understood, although multiple lines of
evidence support a direct role of PS1 in facilitating
-cleavage of
APP, Notch, and other substrates (1, 50). On the other hand, it has
been reported (24) that PS1s may play multiple physiological roles such
as those in calcium homeostasis, neuronal development, neurite
outgrowth, apoptosis, synaptic plasticity, and tumorigenesis. Recent
evidence indicates a novel function of PS1 in regulating intracellular
trafficking of a selected set of proteins including those associated
with PS1 and
APP metabolites (25, 26, 42). In the present report, we
demonstrate the following: 1) PS1 deficiency leads to the accelerated
trafficking of
APP from both the TGN and the ER; 2) loss-of-function
PS1 mutants with impaired
-secretase function increase ER biogenesis of
APP-containing vesicles without affecting TGN budding and eventually elevate the amount of
APP transported to the plasma membrane; 3) FAD-linked PS1 mutants impair
APP trafficking from the
TGN to the plasma membrane, as well as from the ER to Golgi, resulting
in delayed delivery of
APP to the cell surface; 4) a profound
reduction of surface
APP at axonal terminals of neurons that express
FAD-linked PS1 mutants. Taken together, these results indicate that the
role of PS1 in facilitating
-secretase processing of APP extends
beyond its putative function in the catalytic process.
APP metabolism by altering
APP trafficking (Fig. 7). Our model proposes that PS1 provides
a retention signal, which guides
APP delivery to appropriate
compartments where
-secretase processing occurs (Fig.
7a). Gain-of-function FAD mutants (Fig. 7c) may
direct sustained retention of
APP-containing vesicles and
consequently increase the availability of
APP to cleavage enzymes
resident in the TGN and/or ER, the major sites of intracellular A
generation. In contrast, PS1 knockout (Fig. 7b) or
loss-of-function mutants (Fig. 7b') abolish or reduce,
respectively, the retention property and thereby allow accelerated
APP trafficking to the cell surface. In addition, based upon our
observations and another report (42), it is conceivable that PS1
regulates
APP trafficking in both the secretory as well as the
endocytic pathway where some A
may also be generated (51).
View larger version (30K):
[in a new window]
Fig. 7.
Model proposing that PS1 may regulate
APP metabolism by altering
APP trafficking through the secretory pathway.
a, it is proposed that PS1 provides a retention signal to
guide
APP delivery to appropriate compartments where
-secretase
processing occurs. b, in the absence of PS1 expression
APP trafficking is accelerated. As a consequence, A
generation is
reduced. b', PS1 loss-of-function mutants accelerate
APP
transport from the ER without affecting budding from the TGN. The
amount of
APP molecules in each vesicle derived from TGN membrane is
increased without altering the rate of vesicle budding. Therefore,
APP delivery to cell surface is increased. c, in
contrast, FAD-linked PS1 mutants may direct sustained retention of
APP, and consequently increase the availability of
APP to
cleavage enzymes resident in the TGN and/or ER, the major intracellular
sites for A
generation. However, the possibilities that PS1 may
recruit certain cytosolic trafficking factors (such as phospholipase D1
and/or Rab11) to TGN and/or ER membrane and thereby regulate
APP
transport cannot be excluded.
Previously it was shown that PS1 deficiency causes missorting of select
type I membrane proteins. For example, PS1 is required for the
maturation and intracellular trafficking of nicastrin, an integral
component of the -secretase complex (14, 28, 29, 52). Moreover, in
PS1-deficient neurons, telencephalin/ICAM is translocated from the
plasma membrane to large intracellular clusters (26). It is interesting
to note that PS1 deficiency causes enhanced localization of
APP at
the cell surface but has the opposite effect on surface accumulation of
nicastrin and telencephalin/ICAM.
To fully understand the regulation of APP metabolism by PS1, it
would be important to determine whether PS1 exerts an effect on
trafficking of
APP CTFs similar to that on full-length
APP. It
has been shown that loss of PS1 results in the accumulation of the
-/
-CTF (25, 40). However, further studies on the subcellular
distribution of CTFs in PS1-deficient cells indicate the complexity of
intracellular trafficking of CTFs. For example, the generation of CTFs
mostly occurs in the late secretory compartments, whereas
APP CTFs
accumulate in the ER, Golgi, and lysosomes (53). It has also been
reported (53) that CTFs may accumulate in restricted and unpredicted
intracellular compartments in PS1-deficient cells. Although our
preliminary data indicate that PS1 may regulate intracellular trafficking of CTFs in a similar manner as its regulation of
full-length
APP (data not shown), vesicle budding assays
(pulse-chase and low temperature incubations) have not yet
distinguished between trafficking and production of CTFs in the ER and
Golgi compartments. More rigorous studies are underway to establish
appropriate cell lines (e.g. those overexpressing
CTFs)
and optimal experimental conditions.
Much remains to be learned about the mechanisms by which PS1 regulates
intracellular trafficking of APP. Based on our permeabilized cell
data, budding of vesicles containing FGFR1 and NCAM is not influenced by PS1 function, indicating that PS1 exerts its regulation on select membrane proteins. One attractive model is that PS1 regulates
the recruitment or the association of trafficking factors with
cytoplasmic sorting signals within
APP, thereby selectively regulating the sorting of
APP to the surface. In the absence of PS
function,
APP containing vesicles may be transported to the cell
surface via the default constitutive pathway. On the other hand,
FAD-linked mutations modify the interaction between
APP and
trafficking factors in a manner that interferes with efficient
APP
trafficking. In this regard, it has been reported that PS1 and PS2
associate with Rab11, a member of the GTP-binding protein family of
membrane trafficking regulators implicated in protein transport along
the biosynthetic and endocytic pathways (54). In addition, PS1 binds to
Rab GDP dissociation inhibitor (RabGDI), a protein that functions in
vesicular membrane transport to recycle Rab GTPases, and PS1 deficiency
leads to impaired Rab-GDI membrane association (55). Furthermore, our
preliminary data suggest that addition of phospholipase D1 to the
cell-free budding assays prevents impaired
APP trafficking from the
TGN in cells expressing FAD PS1 mutations, whereas inhibition of
phospholipase D1 activity by primary butanol diminishes the accelerated
APP trafficking from the TGN in cells expressing loss-of-function PS1 mutations.
Finally, our studies demonstrate that FAD-linked PS1 mutants result in
a decreased distribution of surface APP in axons and axonal
terminals of neurons. This finding suggests that PS1 may specifically
affect targeting to and/or fusion of
APP-containing vesicles at the
nerve terminus. As reported previously, full-length
APP has been
implicated in a number of physiological functions such as synapse
formation, growth cone outgrowth, and axon guidance (55, 56).
Furthermore, based on the demonstration that
APP plays an essential
role in axonal trafficking (47, 48), our findings that PS1 may regulate
APP sorting along the axon have much broader implications as well.
For example, impaired delivery of full-length
APP to the cell
surface at axonal terminals by FAD-linked PS1 variants may interfere
with neurite initiation, elongation and branching, and synaptic
plasticity. It is important to note that in AD presynaptic pathology is
more severe than neuronal loss (57). By affecting vesicle transport and
surface delivery of full-length
APP, pathogenic PS1 mutants might
directly modulate
APP metabolism and, in addition, indirectly
contribute to the pathogenesis and progression of AD.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants AG09464 (to P. G. and H. X.), AG19070 (to G. T.), AG21495 (to G. T.), AG021494 (to S. S. S.), and NS40039 (to H. Z.), the Alzheimer's Association (to G. T. and H. X.), the Ellison Medical Foundation (to H. X.), and the American Health Assistance Foundation (to G. T. and H. X.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by an Adler Foundation post-doctoral fellowship.
** To whom correspondence should be addressed. Tel.: 212-327-7567; Fax: 212-327-7888; E-mail: xuh@mail.rockefeller.edu.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209065200
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ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
PS, presenilins;
APP,
-amyloid precursor protein;
A
,
-amyloid;
FAD, familial AD;
TGN, trans-Golgi network;
ER, endoplasmic reticulum;
CTFs, C-terminal fragments;
wt, wild type;
FITC, fluorescein isothiocyanate;
mAb, monoclonal antibody;
FGFR1, fibroblast growth factor receptor;
NCAM, neural cell
adhesion molecule.
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