Morphologic and biochemical analysis of the intracellular trafficking of the Alzheimer beta/A4 amyloid precursor protein.

Abstract

Abnormal metabolic processing of the beta/A4 amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease. Several aspects of normal APP processing have been elucidated, but the precise cellular trafficking of APP remains unclear. To investigate APP trafficking pathways further, we have examined the subcellular distribution of APP in rat brain tissue and a variety of cultured cell types, and correlated this distribution with the biochemical processing of APP. In immunofluorescence microscopy of rat brain sections, APP immunoreactivity was concentrated in the Golgi complex and in proximal axon segments. In addition, a lower level of punctate fluorescence was visible throughout the neuropil. By immunoelectron microscopy of rat brain tissue fragments, APP was found associated with Golgi elements and with medium-sized, invaginated vesicles in both axons and dendrites. Prominent localization of APP to the Golgi complex was also found in primary cultures of rat hippocampal neurons and in non-neuronal cell lines. When cultured cells were treated with brefeldin A (BFA), APP immunoreactivity changed from a Golgi-like to an endoplasmic reticulum-like distribution. No APP was detected in the BFA-induced reticulum identified by the transferrin receptor, indicating that concentration of APP in the Golgi does not reflect recycling between the trans-Golgi network and early endosomal system. In immunoblots of BFA-treated cells, there was an accumulation of full-length APP and inhibition of APP secretory processing. Treatment with phorbol ester resulted in a marked elevation of APP secretion, but no obvious redistribution of APP immunoreactivity was apparent at the light microscope level. The lysosomotropic drug chloroquine induced accumulation of APP in cell lysates, as seen by immunoblotting. Immunofluorescence microscopy of chloroquine-treated cells demonstrated a colocalization of APP with the lysosomal marker Igp 120, whereas no colocalization was seen in untreated cells. Taken together, these results support a scheme in which APP is concentrated in the Golgi complex as it travels through the central vacuolar system en route to the plasma membrane for secretion of its amino-terminal domain and/or to lysosomes for degradation.

Full text

Page 1

The Journal of Neuroscience, May 1994, 74(5): 31223138
Morr>holocric and Biochemical
of the Alzbeimer
Analysis
Precursor Protein
of the Intracellular Trafficking
@/A4 Amyloid
Gregg
Pietro De Camilli2~3
L. Caporaso,’ Kohji Takei,2v3 Samuel E. Gandy, Is4 Michela Matteoli,3 Olaf Mundigl,3 Paul Greengard,’ and
‘Laboratory
Hughes Medical Institute and 3Department
Medicine, New Haven, Connecticut
Hospital-Cornell Medical Center, New York, New York 10021
of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, “Howard
of Cell Biology, Yale University School of Medicine, Boyer Center for Molecular
06536, and 4Department of Neurology and Neuroscience, The New York
Abnormal
cursor
esis
processing
trafficking
ficking
distribution
cell types,
ical processing
In immunofluorescence
APP immunoreactivity
plex and
level of punctate
neuropil. By immunoelectron
fragments, APP
and with medium-sized,
and dendrites.
complex was also
campal neurons
When cultured
APP immunoreactivity
doplasmic reticulum-like
in the BFA-induced
receptor, indicating
does not reflect
and early endosomal
cells, there was an accumulation
hibition of APP secretory
ester resulted
no obvious redistribution
metabolic processing
has been
disease.
been elucidated,
of the B/A4
implicated
Several aspects
but the precise
unclear. To investigate
we have examined
and a variety
this distribution
amyloid pre-
protein
of Alzheimer
(APP) in the pathogen-
of normal APP
have
of APP remains
pathways
of APP in rat brain tissue
and correlated
of APP.
cellular
APP traf-
further, the subcellular
of cultured
with the biochem-
microscopy
was concentrated
axon segments.
fluorescence
of rat brain
in the Golgi
In addition,
was visible
microscopy
associated with Golgi
invaginated vesicles
localization
in primary cultures
and in non-neuronal
cells were treated
changed from
distribution.
reticulum identified
that concentration
recycling between
system. In immunoblots
of full-length
processing.
in a marked elevation
of APP immunoreactivity
sections,
com-
in proximal a lower
throughout
of rat brain
the
tissue
was found elements
axons
to the
of rat hippo-
in both
Prominent of APP Golgi
found
cell lines.
with brefeldin
a Golgi-like
No APP was detected
by the transferrin
of APP
the frans-Golgi
of BFA-treated
A (BFA),
to an en-
in the Golgi
network
APP and in-
with phorbol
secretion,
was ap-
Treatment
of APP but
Received June 28, 1993; revised Oct. 15, 1993; accepted Nov. 1, 1993.
We thank Drs. 0. da Cruz e Silva and K. Iverfeldt for kindly providing COS
cells, R. Cofiell for preparing rat brain sections, E. Griggs for providing photo-
graphic assistance, and Drs. M. Solimena and H. Stukenbrok for thoughtful dis-
cussions. We also thank Drs. K. Beyreuther, D. Bole, K. Buckley, I. Mellman, K.
Moreman. I. Sandoval. J. Saraste. D. Selkoe. and I. Trowbridae
viding antibodies. This-research was supported by U.S. Public Health S&vice
Medical Scientist Training Program Grant GM-07739 (G.L.C.), U.S. Public Health
Service Grant AG- 11508 and a Cornell Scholar Award in the Biomedical Sciences
(S.E.G.), a long-term European Molecular Biology Organization fellowship (M.M.),
U.S. Public Health Service Grants AG-09464 and AG-1049 1 (P.G.), a M&night
Endowment for the Neurosciences, and National Institute of Mental Health Grant
MH 45191 (P.D.C.).
Correspondence should be addressed to Paul Greengard, Ph.D., Laboratory of
Molecular and Cellular Neuroscience, The Rockefeller University,
Avenue, New York, NY 1002 1.
Copyright 0 I994 Society for Neuroscience
for kindly pro-
1230 York
0270-6474/94/l 43 122-17$05.00/O
parent
drug
ates,
croscopy
localization
as no colocalization
together,
concentrated
central vacuolar
for secretion
somes for degradation.
[Key words:
A, chloroquine,
at the
chloroquine
as seen
light microscope
induced
by immunoblotting.
of chloroquine-treated
of APP with the lysosomal
was seen
these results
in the Golgi
system
of its amino-terminal
level. The lysosomotropic
of APP
lmmunofluorescence
cells demonstrated
marker
in untreated
a scheme in which
as it travels
to the plasma
domain
accumulation in cell lys-
mi-
a co-
where-
Taken
APP
Igpl20,
cells.
support
complex
en route
is
through
membrane
and/or
the
to lyso-
Golgi
lysosomes,
complex, synaptic vesicles, brefeldin
phorbol ester]
The extracellular deposition of PIA4 amyloid peptide in brain
cortical parenchyma and cerebromeningeal
pathological hallmark ofAlzheimer
1984; Tomlinson and Corsellis, 1984; Masters et al., 1985). The
PIA4 peptide arises from proteolytic
precursor protein (APP), a transmembrane
exists as multiple isoforms resulting from alternative
of a single transcript (Goldgaber et al., 1987; Kang et al., 1987;
Robakis et al., 1987; Tanzi et al., 1987, 1988; Kitaguchi et al.,
1988; Ponte et al., 1988). It is believed that abnormalities
the APP molecule or in its metabolism can give rise to Alzhei-
mer disease. Several mutations in the APP gene have been iden-
tified in the kindred of patients suffering from early-onset, her-
itable forms of the disease (Chattier-Harlin
et al., 199 1; Murrell et al., 199 1; Naruse et al., 199 1; Mullan et
al., 1992). Mutations in the APP gene are also apparently re-
sponsible for hereditary cerebral hemorrhage with amyloidosis
(Dutch type), a rare inherited disorder in which deposition of
PIA4 peptide in cerebral blood vessels produces fatal hemor-
rhages (Levy et al., 1990; Hendriks et al., 1992). Consequently,
elucidation of the normal metabolic pathways of APP should
provide a base for studying the processing abnormalities
volved in the pathogenesis of these diseases.
Current evidence suggests the existence of multiple processing
pathways for APP. One pool of APP molecules is cleaved within
the PIA4 region, with the APP ectodomain secreted into cere-
brospinal fluid and into the medium of cultured cells (Palmert
et al., 1989; Weidemann et al., 1989; Oltersdorf et al., 1990).
Since the proteolytic event that results in APP secretion occurs
within the PIA4 domain of APP, it precludes a contribution
this population of molecules to amyloid deposits (Esch et al.,
blood vessels is a
disease (Glenner and Wong,
cleavage of the amyloid
glycoprotein that
splicing
in
et al., 199 1; Goate
in-
of

Page 2

The Journal of Neuroscience, May 1994, 14(5) 3123
1990; Sisodia et al., 1990). The principal cellular site of APP
secretory cleavage has not been determined, but recently it has
been shown that at least some molecules may be cleaved at the
cell surface (Sisodia, 1992). Although the kinetics of APP ec-
todomain secretion indicate that APP does not follow the stan-
dard regulated secretory pathway (Overly et al., 1991) APP
secretion can be stimulated either by direct activation of protein
kinase C with phorbol esters (Caporaso et al., 1992b; Gillespie
et al., 1992; S. Sinha, personal communication)
of first messengers that act through protein kinase C (Buxbaum
et al., 1992; Nitsch et al., 1992), suggesting a regulatory mech-
anism for this processing pathway. The mechanism by which
protein kinase C modulates secretion of APP ectodomain
not been determined, although protein kinase C can phospho-
rylate APP in vitro (Gandy et al., 1988; Suzuki et al., 1992).
In most cell types that have been studied, the secretory path-
way accounts for only a small fraction of total APP processing
(Weidemann et al., 1989; Haass et al., 1991; Caporaso et al.,
1992a; Knops et al., 1992). In rat neuroendocrine
the majority of APP molecules are proteolytically
a chloroquine-sensitive intracellular
endosomes and/or lysosomes (Caporaso et al., 1992a). It has
also been shown that full-length APP and APP degradative frag-
ments are present in a lysosome-enriched
(Haass et ai., 1992a), that potentially amyloidogenic
ments are probably processed in endosomes and lysosomes
(Golde et al., 1992; Knops et al., 1992), and that APP immu-
noreactivity is present in neuronal structures identified as sec-
ondary lysosomes (Benowitz et al., 1989). Furthermore,
length APP and the carboxyl-terminal
from secretory cleavage are enriched in clathrin-coated
suggesting that these protein species are targeted to the endo-
somal system after endocytosis from the plasma membrane or
directly from the trans-Golgi network (TGN) (Nordstedt et al.,
1993). It was demonstrated recently that intact @A4 peptide is
released from cultured cells and is present in plasma and ce-
rebrospinal fluid (Haass et al., 1992b; Seubert et al., 1992), and
that production of secreted P/A4 might occur in the endosomal/
lysosomal system (Shoji et al., 1992) additionally
importance of this compartment
In the present study, the subcellular distribution
examined in a variety of cell types in an attempt to elucidate
APP trafficking further. Employing
vidual cellular compartments and pharmacological agents known
to affect organelle function or signal transduction,
the localization of APP with the biochemical processing of the
molecule.
or by application
has
PC12 cells,
degraded in
presumably compartment,
subcellular fraction
APP frag-
full-
APP fragment resulting
vesicles,
stressing the
in APP metabolism.
of APP was
markers specific for indi-
we correlate
Materials
Antibodies.
nal APP antibody
tibody identifies
forms as well as carboxyl-terminal
1990). [It is possible that antibody
es, cross-react with other proteins
Wasco et al., 1992, or APLP2, Wasco et al., 1993). Experiments
to clarify this issue are underway.] Other antibodies
as follows: a monoclonal antibody
(22Cll; Dr. Konrad Beyreuther,
Germany), polyclonal rabbit serum raised against the carboxyl terminus
of APP (C7; Dr. Dennis Selkoe, Harvard
a monoclonal antibody that recognizes an integral membrane
ofthe truns-Golgi and TGN (GIMP,;
Autonoma de Madrid, Cantoblanco,
tibody against the synaptic vesicle protein SV2 (Dr. Kathleen
and Methods
Preparation
(369A) has been described previously,
immature and mature holomolecules
of affinity-purified rabbit anti-carboxyl-termi-
and this an-
of all APP iso-
(Buxbaum APP fragments
369A may, under some circumstanc-
in the APP family
et al.,
(e.g., APLPl,
designed
were generous gifts,
terminus
of Heidelberg,
against the amino
University
of APP
Heidelberg,
Medical School, Boston, MA),
protein
Dr. Ignacio Sandoval, Universidad
Madrid, Spain), a monoclonal an-
Buckley,
Harvard
against mannosidase
of Technology,
the intermediate
Institute for Cancer Research, Stockholm,
tibody against the cytoplasmic
(Tf-R) (Dr. Ian Trowbridge,
clonal antibody that recognizes the endoplasmic
BiP (Dr. David Bole, University
monoclonal antibody against the lysosomal marker lgp 120 (Dr. Ira Mell-
man, Yale University School of Medicine,
FITC-conjugated goat anti-mouse
Chemical Co. (St. Louis, MO), and rhodamine-conjugated
rabbit antibody was obtained from Boehringer
(Indianapolis, IN). FITC-conjugated
obtained from Vector Laboratories,
jugated lentil lectin was obtained from E-Y Laboratories (San Mateo,
CA). Protein A-gold particles were prepared according to the method
of Slot and Geuze (1985). Donkey
horseradish peroxidase-coupled secondary antibodies
from Amersham Corp. (Arlington Heights, IL).
Cell cultures. Primary cultures of rat hippocampal
pared from l&d-old fetal rats as described (Banker and Cowan, 1977;
Bartlett and Banker, 1984). Cells were arown for 4 d on oolv-r-lvsine-
treated glass coverslips in’minimum
containing 1% HLI supplement (Ventrex, Portland,
mine, and 1 mg/ml BSA.
Chinese hamster ovary (CHO) cells were grown in Dulbecco’s
ified Eagle’s medium (DMEM) containing
34 pg/rnl proline, 100 U/ml penicillin,
African green monkey kidney (COS) cells were grown in DMEM
taining 10% heat-inactivated FBS, 20 mM HEPES
antibiotic/antimycotic solution (GIBCO-Bethesda
Gaithersburg, MD). Undifferentiated
cells were grown in DMEM containing
heat-inactivated horse serum, and 1 x antibiotic/antimycotic
Rat insulinoma (RINmSF) cells (Gazdar et al., 1980) were grown in
RPM1 1640 medium containing 10% fetal calf serum. Mouse insuli-
noma (flTC3) cells (Efrat et al.. 1988) were arown in Click medium
(Irvine”Scientific, Santa Ana, CA) containing
mary cultures and all cell lines were grown in 5% CO, at 37°C.
Drug treatments. Brefeldin A (BFA) was obtained
Technologies (Madison, WI) and was stored at - 20°C as a 1000 x stock
solution of 10 mg/ml in methanol.
was obtained from LC Services (Wobum,
as a 1000x stock solution of 1 rnr.4 in 50% dimethvl
roquine was obtained from Sigma Chemical
as a 1000x stock solution of 50 mM. Cells were incubated
indicated times in medium containing
fixation for immunocytochemistry
analysis.
Immunojluorescence labeling. Cells were grown for 2-3 d on poly-L-
omithine-coated glass coverslips. Following incubation
or in medium containing drugs, cells were washed once with 120 mM
phosphate buffer (pH 7.4) and then fixed for 30 min at 37°C with 4%
formaldehyde (freshly prepared from paraformaldehyde),
120 mM phosphate buffer. All subsequent steps were performed at room
temperature. After a wash with high-salt
phosphate buffer), cells were incubated
dilution buffer” (GSDB; 0.45 M NaCl, 20 mM phosphate buffer, 0.3%
Triton X-100, 17% goat serum) in order to permeabilize
and block nonspecific secondary antibody
incubated for 2-3 hr with primary antibodies
lowing washes with high-salt PBS, cells were incubated
with fluorescent secondary antibodies
washed with high-salt PBS and then briefly with 5 mM phosphate buffer
before mounting the coverslips onto glass slides using freshly prepared
mounting solution [70% glycerol, 1 mg/ml
Chemical Co.), 150 mM NaCl, 10 mM phosphate
viewed with a Zeiss Axiophot microscope equipped with epifluorescence
optics and photographed with Kodak TMAX-100
Brains of adult Sprague-Dawley
perfusion with 4% paraformaldehyde
7 pm sections were prepared (De Camilli
were labeled for immunofluorescence
scribed above.
Medical School, Boston, MA), a rabbit polyclonal
II (Dr. Kelley Moreman,
Cambridge, MA), a rabbit polyclonal
compartment protein ~58 (Dr. Jaakko Saraste, Ludwig
antibody
Institute
against
Massachusetts
antibody
Sweden), a monoclonal
of human transferrin
Salk Institute, San Diego, CA), a mono-
reticulum
of Michigan, Ann Arbor, MI), and a
an-
domain receptor
(ER) marker
New Haven, CT).
antibody was obtained from Sigma
goat anti-
Biochemicals
(WGA) was
CA). FITC-con-
Mannheim
wheat germ agglutinin
Inc. (Burlingame,
anti-rabbit and sheep anti-mouse
were purchased
neurons were pre-
essential medium without serum,
ME), 2 mM gluta-
mod-
FBS, 10% heat-inactivated
and 100 &ml streptomycin.
con-
(pH 7.4) and 1 x
Research Labs,
(PC12)
FBS, 5%
solution.
rat pheochromocytoma
10% heat-inactivated
3% fetal calf serum. Pri-
from Epicentre
Phorbol 12,13-dibutyrate
MA) and was stored at -20°C
sulfoxide.
Co. and was stored at 4°C
(PDBu)
Chlo-
for the
drug, and then washed prior to
or prior to lysis for immunoblot
in medium alone
4% sucrose in
PBS (500 mM NaCl, 20 mM
for 30 min with “goat serum
membranes
binding sites. Cells were next
prepared in GSDB. Fol-
for 30-90 min
prepared in GSDB. Cells were
p-phenylenediamine (Sigma
buffer]. Slides were
film.
gm) were fixed by rats (150-200
in 120 mM phosphate buffer and
et al., 1983a). Brain sections
microscopy essentially as de-

Page 3

3124 Caporaso et al. * Trafficking of APP
Figure 1.
lular localization
rat brain by immunofluorescence
croscopy. Rat brain sections were dou-
ble labeled for APP (antibody
(u-d) and GIMP, (a’-&). The following
populations of cells are shown: a, a’,
cortical forebrain pyramidal
hippocampal pyramidal
brainstem neurons; and d, d’, cerebellar
Purkinje cells. APP immunoreactivity
in the proximal segment of axons is in-
dicated (arrows). Scale Bars, 20 pm.
Comparison of the subcel-
of APP and GIMP, in
mi-
369A)
cells; b, b’,
cells; c, c’,
APP GlMPt
APP
GlMPt
GlMPt
Immunoelectron
brain tissue fragments and immunoe~ectron
as described nreviouslv
Immunobh unuZys~~‘Cells
dishes. Following drug treatment,
microscopy. Preparation of agarose-embedded
microscopy were performed
et al., 1983b: Takei et al., 1992).
were grown for 2-3 d on 6 cm culture
the medium was removed,
rat
were washed with 120 mM phosphate buffer or Hank’s balanced saline
solution and scraped from the dishes in 400-600 ~1 of 1% SDS, and the
lvsates were boiled and sonicated.
x g for 10 min, supematants were assayed for total protein using the
BCA system (Pierce, Rockford, IL). Aliquots
(De Camilli
the cells
Following centrifuaation at 14.000
of cell lysates containing

Page 4

The Journal of Neuroscience, May 1994, 14(5) 3125
equal amounts
lysate protein, were diluted
SDS-pblyacrylamide
(Laemmli. 1970). and transferred to nitrocellulose
et al., 1979). The procedure
elsewhere (Nordstedt et al., 1993). Immunoreactivity
with the enhanced chemiluminescence
cording to the manufacturer’s
x-ray film.
Pulse-chase labeling and immunoprecipitation. Experimental
dures for %-methionine pulse labeling
cipitation using antibody 369A were essentially as described previously
(Caporaso et al., 1992a). When APP maturation
munoprecipitates were treated with endoglycosidase
ringerMannheim Biochemicals) for 16 hror with neuraminidase
rineer Mannheim Biochemicals) for 2 hr at 37°C as described (Teixido
----0-m
and Massague, 1988). Control samples were treated with enzyme buffer
alone for 16 hr at 37°C. Incubations
5 x gel sample buffer and boiling.
of protein, or volumes
and boiled in sample buffer, separated on
gels by electrophoresis under reducing conditions
of medium normalized for cell
membranes
has been described
was visualized
system (Amersham
instructions, and blots were exposed to
(Towbin
for immunoblotting
Corp.) ac-
proce-
of CHO cells and immunopre-
was examined,
H (endo H) (Boeh-
im-
(Boeh-
~~
were stopped by the addition of
Results
APP is concentrated in the Go& complex of neurons and in
vesicular structures of dendrites and axons
To examine
the subcellular localization of APP, rat brains were
fixed by perfusion with formaldehyde
pm slices were examined by immunofluorescence
(De Camilli et al., 1983a) with an antibody against the carboxyl
terminus of APP (369A; Buxbaum et al., 1990). In the brain
regions studied-forebrain, hippocampus,
ebellum- most neurons were intensely APP immunoreactive
(Fig. 1). Some APP immunoreactivity
variety of supporting cells, including glial cells. APP was ob-
served predominantly in perinuclear structures in all cell types.
In neurons, this intense immunoreactivity
proximal portions of cell processes.
This distribution was suggestive of a concentration
in the region of the neuronal Golgi complex (De Camilli et al.,
1986). Brain sections were therefore double labeled with an
antibody directed against GIMP,, an integral membrane protein
localized to the trans-Golgi and TGN (Yuan et al., 1987). Cel-
lular areas of the greatest APP immunoreactivity
GIMP,-immunoreactive areas in perikarya and dendrites (Fig.
1). In addition, high levels of APP staining were found in the
proximal portion of axons, which do not contain elements of
the Golgi complex (De Camilli
accordingly negative for GIMP,
la,a’,b,b$ A high-power view of a cerebellar Purkinje cell with
its initial axonal segment is shown in Figure 1, d and d’. APP
immunoreactivity extended along the axon into the granule cell
layer (Fig. Id). This staining became weaker with increasing
distance from the perikaryon. Diffuse punctate APP immuno-
fluorescence was present throughout
background immunofluorescence
d?, but no accumulation of APP was seen at synapses.
A predominant localization of APP in the region of the Golgi
complex was also observed in neurons in culture (Banker and
Cowan, 1977; Bartlett and Banker, 1984) (Fig. 2a). In addition,
in these cells a moderate to intense APP immunoreactivity
present as fine puncta in neuronal processes. APP was not con-
centrated at synaptic vesicles, however, as indicated by double
labeling for the synaptic vesicle protein SV2 (Buckley and Kelly,
1985) (Fig. 2b).
To confirm the Golgi localization of APP and to examine the
nature of the APP immunoreactivity
fragments of brain tissue were obtained by coarse homogeni-
and cryosectioned, and 7
microscopy
brainstem, and cer-
was also detected in a
extended into the
of APP
coincided with
et al., 1986) and which were
immunoreactivity (Fig.
the neuropil (compare the
of Fig. 1, a-d, with that of a 1
was
seen in neuronal processes,
APP
Figure 2. Localization of APP and SV2 in primary cultures of rat
hippocampal neurons by immunofluorescence
pal neurons were grown in culture for 4 d before fixation and double
labeled for APP (antibody 369A) (a) and SV2 (b). Scale bar, 20 pm.
microscopy. Hippocam-
zation, embedded in agarose, immunogold
finally Epon embedded (De Camilli et al., 1983b; Takei et al.,
1992). In fragments of the Golgi complex, the presence of gold
particles on cistemae was visible (Fig. 3a). This labeling of the
Golgi complex was not seen in control preparations reacted with
preimmune serum.
In microscopic fields containing fragments of cell processes
and synapses, APP immunoreactivity
ated with medium-sized vesicles, many of which were charac-
terized by outer and inner membranes, the latter appearing to
arise by the invagination of the outer membrane (Fig. 3&f).
Gold particles were found at the cytoplasmic face of the outer
membrane of these structures. The vesicles decorated with gold
particles were seen both in myelinated
myelinated (Fig. 3d) axons as well as in pre- and postsynaptic
compartments (Fig. 3e,f). No other dendritic or axonal struc-
tures were labeled, nor were the majority of synaptic vesicles
(Fig. 3ef).
labeled for APP, and
was found to be associ-
(Fig. 3b,c) and in un-
Golgi localization of APP in cultured non-neuronal
Since we wished to determine the effects of experimental
nipulations on the subcellular distribution
cells
ma-
ofAPP, several mam-

Page 5

3126 Caporaso et al. * Trafficking of APP
Figure 3. Localization of APP in rat
brain tissue fragments by immunoelec-
tron microscopy. Rat brain tissue frag-
ments from forebrain (a, b, d) or from
cerebellum plus brainstem (c, e, j) were
embedded in agarose and probed with
antibody 369A and colloidal gold-con-
jugated protein A prior to plastic em-
bedding. APP immunoreactivity, as re-
vealed by gold particle accumulation,
is visible on Golgi membranes (small
arrows, a) and on medium-sized vesi-
cles (large arrows and b, inset) in cell
processes (b, c, d), including myelinated
(b, c) axons, and in pre- (e) and post-
synaptic compartments (fl. Some of
these vesicles contain an internal mem-
brane that appears to originate by an
invagination of the outer membrane. A
mitochondrion is indicated in a (M).
Scale bars, 200 nm.
malian cell lines were examined to select a cell type appropriate
for immunofluorescence microscopy of APP. Mouse insulinoma
@TC3), rat insulinoma (RINmSF),
(PC1 2), African green monkey kidney (COS), and Chinese ham-
ster ovary (CHO) cells all exhibited a predominant
tion of APP at a juxtanuclear
ing seen in the cytoplasm (Fig. 4). The juxtanuclear
coincided with the localization of the Golgi complex and pri-
concentra-
location, with fine punctate stain-
rat pheochromocytoma staining