J Biol Chem, Vol. 274, Issue 45, 32301-32308, November 5, 1999
Uptake, Degradation, and Release of Fibrillar and Soluble Forms
of Alzheimer's Amyloid 
-Peptide by Microglial Cells*
Haeyong
Chung
§,
Melanie I.
Brazil§,
Thwe Thwe
Soe§, and
Frederick R.
Maxfield§¶
From the § Department of Biochemistry, Weill Medical
College of Cornell University, New York, New York 10021 and the
Department of Pathology, Columbia University College of
Physicians and Surgeons, New York, New York 10032
 |
ABSTRACT |
Microglia are phagocytic cells that are the main
inflammatory response cells of the central nervous system. In
Alzheimer's disease brain, activated microglia are concentrated in
regions of compact amyloid deposits that contain the 39-43-amino acid A
peptide. We examined the uptake, degradation, and release of small
aggregates of fibrillar A (fA) or soluble A (sA) by microglia. We found that although some degradation of fA was observed over 3 days, no further degradation was observed over the next
9 days. Instead, there was a slow release of intact A. The poor
degradation was not due to inhibition of lysosomal function, since the
rate of 
2-macroglobulin degradation was not affected by the presence
of fA in the late endosomes/lysosomes. In contrast to fA,
internalization of sA was not saturable. After internalization, sA was released rapidly from microglia, and very little was
degraded. These data show that fA and sA interact differently
with microglia but that after internalization a large fraction of both
are released without degradation.
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INTRODUCTION |
Alzheimer's disease
(AD)1 represents the most
frequent cause of dementia in the elderly, accounting for more than
half of all cases (1). The characteristic histological features of AD
are senile plaques, neurofibrillary tangles, congophilic angiopathy of
the vessels, neuropil threads, and neuronal cell loss (1, 2). Senile
plaques are classified into two major types: the classical
(neuritic) and the diffuse (preamyloid) plaques. The classical plaque
is a complex lesion of the cortical neuropil containing several
abnormal elements: a central deposit of extracellular amyloid fibrils
("the core" composed of -amyloid or A peptide (3, 4))
surrounded by dystrophic neurites (both dendrites and axonal
terminals), activated microglia, and reactive astrocytes (1, 2). The
diffuse plaques are devoid of amyloid core, with very few or no
surrounding dystrophic neurites or glia and contain nonfibrillar
(amorphous) A. Despite intense investigation, the sequence of events
that lead to neuronal pathology in AD is still largely unknown. Many
studies have supported the hypothesis that A deposition may play an
early and, in some cases, causative role in the disease.
A is a 39-43-amino acid fragment of a larger membrane-spanning
glycoprotein called -amyloid precursor protein (APP) (3, 5). The
different forms of A have varying solubilities. A is
constitutively produced in cultured cells (6, 7), and soluble A
(sA) is present at similar concentrations (10
8 to
1010 M) in normal and AD cerebrospinal fluid
(7, 8). These observations indicate that A production is a normal
physiological process in vivo and in vitro. sA
can spontaneously form insoluble assemblies of -pleated sheet
(fibrillar) conformation similar to amyloid fibrils found in AD brain.
The formation of fibrils in vitro depends on concentration,
time, pH, temperature, and ionic strength (9-13).
The A that accumulates in AD brains is heterogeneous at its C
terminus, resulting in peptides 39-43 amino acids in length. It has
been shown that diffuse plaques almost exclusively consist of
A-(1-42/43) (14). In contrast, neuritic plaques contain both
A-(1-40) (A40) and A-(1-42) (A42) and also shorter A peptides that are truncated at the N terminus (14-17). A40 is believed to be the major isoform generated by cultured cells (8, 18,
19) as well as in cerebrospinal fluid (20), while A42 is the
predominant species accumulated in senile plaques in AD brains as well
as in the brains of nondemented aged individuals and appears to be the
initially deposited species (14, 16, 21). Differences in the deposited
and soluble forms of A may be significant in -amyloidogenesis.
In vitro, A42 is less soluble and forms fibrils at a
greater rate than shorter isoforms (11).
Since AD involves the net accumulation of A, it is important to
understand both the synthesis and clearance of A. It has been long
recognized that reactive microglia are closely associated with neuritic
plaques in AD, and these cells could be involved in either the removal
or the production of amyloid fibrils (22). Microglia are
macrophage-like phagocytic cells. They are the main inflammatory
response cells of the brain. Microglia found in normal adult brain are
highly ramified, quiescent cells that retract their processes and
become reactive during CNS injury (22, 23). In AD, quantitative
histopathology has been used to show that clusters of reactive
microglia are frequently associated with the amyloid core and/or within
the plaques, whereas diffuse plaques are generally not associated with
microglia (24-26). Reactive microglia are also found associated with
plaques that develop in a transgenic mouse model expressing mutant
APP (27-29). These observations suggest that brain inflammatory
responses may be directed specifically against the constituents of
neuritic plaques and that one pathway for A-induced neuron damage
may involve inflammatory microglia. Activated microglia are capable of
releasing cytotoxic agents such as proteolytic enzymes, cytokines,
complement proteins, reactive oxygen intermediates, and nitric oxide
(30). Although these considerations suggest that the involvement of
microglia in AD is mainly harmful, these cells could also play a
protective role by digesting amyloid fibrils. It has been suggested
that clearance of amyloid deposits by immunization against A in a
mouse model for AD may occur via microglial cells (31).
While amyloid is present in the intracellular organelles of microglia
(32, 33), it is not known if this results from phagocytosis of
previously deposited amyloid by microglia or from microglial processing
of APP to A. In culture, microglia can synthesize APP
(34-37). Also, some investigators have observed amyloid precursor protein mRNA in microglia in AD brain (38), but others have not
(39, 40). It is likely that some, if not all, of the intracellular A
observed in microglia is extracellular in origin. It is possible that
microglia facilitate the formation of plaques upon ingestion of
fibrillar or soluble A and delivery to acidic endosomes.
We and others have identified receptors on microglia that bind purified
A (41-43). These include members of the scavenger receptor family
(41, 42). We have found that two forms of the scavenger receptor, SRA
and SRB, could mediate the uptake of fA in transfected Chinese
hamster ovary cells (41). We examined the degradation of fA up to 3 days after internalization, both immunocytochemically and
biochemically, and found that microglia showed intracellular
accumulation of fA and slow, incomplete degradation of fA (44).
If microglia do not degrade fA after a certain time point, this
might mean that microglia accumulate fA and participate in the
formation of amyloid. The existence of undegraded fA could be due to
slow degradation of fA in all cells or to a population of cells that
cannot degrade fA. To distinguish between these possibilities, we
followed the degradation for a longer period of time. Since soluble
A production is a normal physiological process, we also studied the
uptake and degradation of sA, and we found that the uptake and
degradation kinetics of fibrillar A were quite different from those
of soluble A. In the present study, we measured the degradation of
fA over the course of 12 days. After a short pulse, there was a
slow, partial degradation for 3 days. After that time, degradation
stopped, but the release of trichloroacetic acid-insoluble material
from microglia continued throughout the 12 days. The degradative
function of late endosomes/lysosomes was not affected by the presence
of fA in these compartments. In contrast to fA, sA was not
internalized by scavenger receptors in microglia, and very little
degradation was observed.
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EXPERIMENTAL PROCEDURES |
Isolation of Microglia--
Microglia were isolated from mixed
glial cultures prepared from newborn mice as described previously (41,
44). The mixed glial cultures were grown in bicarbonate-buffered
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin (Life Technologies, Inc.) at 37 °C in a 5%
CO2 humidified air atmosphere. Microglia were harvested
by orbital shaking for 20-45 min at 150 × g in growth
medium. Cells were centrifuged for 10 min at 350 × g
and plated onto 24-well tissue culture plates at 105
cells/well or onto 35-mm diameter plastic tissue culture dishes in
which a 7-mm diameter hole was punched in the bottom, and a polylysine-coated number 1 glass coverslip was attached beneath the
hole (45) at a density of 104 to 105
cells/coverslip. Cell viability was assayed with a commercial kit
(LIVE/DEAD; Molecular Probes, Inc., Eugene, OR).
Proteins--
1-40 and 1-42 A peptides were purchased from
Bachem (Torrance, CA). 125I-Labeled A was prepared using
the chloramine T method (41, 46). Purified
2-macroglobulin (2M) was converted to the
receptor-binding form by dialyzing in 25 mM methylamine (pH
8.0) for 4 h at room temperature, followed by dialysis against
phosphate-buffered saline for 3 days with four buffer changes at
4 °C. This procedure cleaves the internal thioester bond and
converts 2M to a high affinity ligand for its receptor
(47). 125I-Labeled 2M was prepared using the
chloramine T method (46, 48). Iodinated A and 2M
retain the ability to bind specifically to their receptors (41, 46,
49). The specific activities of 125I-labeled A40,
A42, and 2M in the representative experiments shown
in the figures ranged between 1.2 × 103 and
3.4 × 103 µCi/ng.
Fluorescently labeled A was prepared by conjugation with Cy3, a
carbocyanine dye (Biological Detection Systems Inc., Pittsburgh, PA),
as described previously (41, 44). 2M was conjugated to
fluorescein isothiocyanate (FITC) as described previously (50).
For all of our studies on uptake and degradation of labeled and
unlabeled fA, A was preaggregated before being added to microglia. A was first diluted in labeling medium (Dulbecco's modified Eagle's medium with 10 mg/ml bovine serum albumin) or Dulbecco's modified Eagle's medium with 10% fetal bovine serum and
vortexed. The mixture was then pH-adjusted to 5.0 with 1 M HCl and incubated at 37 °C for 4-16 h. For studies using Cy3 and 125I-labeled soluble A, A was diluted in labeling
medium with 10 mg/ml bovine serum albumin, slowly mixed by pipetting,
and then subjected to ultracentrifugation at 100,000 × g for 1 h at 25 °C. Only the resulting supernatant
was added to microglia to ensure that no aggregated A was added to
the cells. Since A is known to spontaneously aggregate in
vitro, for each experiment using 125I-labeled sA,
we incubated the supernatant as above in 37 °C in a Petri dish in
parallel with the experimental dishes for the same amount of time as
the pulse time. The mixture in the Petri dish was centrifuged at
100,000 × g for 1 h at 4 °C. We found that all
of the 125I-labeled sA remained soluble.
Uptake and Degradation of 125I-Labeled
Ligands--
Cells were grown in 24-well plates for 1-2 days before
the start of the experiment. The cells were washed twice with labeling medium and then incubated with the radiolabeled fibrillar or soluble peptides for 1 h at 37 °C in a 5% CO2 humidified
air atmosphere. The cells were rinsed three times with labeling medium
and then incubated at 37 °C for 15 min to allow the release of
nonspecifically bound A. The cells were rinsed further, twice with
medium without serum and three times with chase medium, which was the
microglia growth medium, and then incubated with 1 ml of chase medium
for varying times. At the end of the chase, the medium bathing the cells was removed, and the radioactivity was measured. The cells were
rinsed and solubilized with 1 M NaOH. The chase medium was precipitated with 10% trichloroacetic acid on ice for 1 h and centrifuged at 14,000 × g for 10 min to separate the
trichloroacetic acid-soluble fraction from the insoluble fraction.
Specific binding was determined by adding unlabeled Ac-LDL, fucoidan,
or 2M as competitive inhibitors at a molar concentration
100-200-fold greater than the radiolabeled ligands. Where indicated,
the excess unlabeled ligands were added to the incubation medium along
with the radiolabeled peptides and were maintained in the labeling
medium for the entire incubation. The radioactivity of these background
dishes was subtracted from the values for the control experiments. The
binding of 125I-labeled fA in the presence of excess
Ac-LDL or fucoidan was less than 10% of control binding, whereas
2M uptake in the presence of excess 2M
was about 25%. All radioactive experiments were conducted with three
wells per condition and repeated at least five times on separate days.
Degradation of Cy3-fA--
Cy3-A (1 µg/ml) was diluted
in pH 7.4 labeling medium, allowed to aggregate at 37 °C for 4 h, and then added to microglia in coverslip dishes. After 1 h of
incubation, cells were washed extensively, three times in labeling
medium, twice in medium without serum, and three times in chase medium.
The cells were incubated in chase medium with no added peptides and
chased for varying times. After chase, cells were rinsed twice with
medium 1 (150 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 5 mM KCl, 1 mM MgCl2, pH 7.4) and fixed with 3.3%
paraformaldehyde diluted in medium 1 for 10 min at room temperature.
Degradation of FITC-2M--
Microglia plated on
coverslip dishes were loaded with Cy3-labeled fA42 (3 µg/ml)
diluted in growth medium for 3 days. Cells were rinsed twice with
labeling medium and pulsed with FITC-2M (5 µg/ml) in
labeling medium for 45 min. Cells were washed extensively as described
previously and then chased for 2, 3, and 6 h. After each chase
time, cells were rinsed with medium 1 and fixed with 3.3%
paraformaldehyde. 20 mM methylamine in medium 1 was added to cells to collapse intracellular pH gradients. Cells were imaged by
confocal microscopy.
Quantitative Fluorescence Microscopy--
Fluorescence
microscopy and digital image collection were performed using a Leica
DMIRB microscope (Leica Mikroscopie und Systeme GmbH, Germany) equipped
with a Princeton Instruments cooled CCD camera driven by
Image-1/MetaMorph Imaging System software (Universal Imaging
Corporation) as described previously (51, 52). Fluorescence
quantification was carried out with a × 25, NA 0.75 objective to
obtain a large number of cells per field, whereas the images for
visualization purposes were obtained with a × 63, NA 1.32 objective (51-55). Fields used for quantification were selected at
random throughout the dish and focused using phase contrast optics
before viewing the fluorescence. Digital fluorescence images were
obtained, and the background signal was removed from the images as
described previously (41, 44, 56). Cells were identified in the image
using an intensity threshold set at the mean pixel intensity for the
entire image plus the S.D. of the pixel intensity. Objects of the size
of cells and above the threshold intensity were identified using
MetaMorph software routines (Universal Imaging, West Chester, PA).
Total fluorescence power per cell was taken as the sum of all intensity in each object identified as a cell. The total fluorescence power per
cell remaining after various chase times was determined for Cy3-fA.
Fluorescent beads were used to normalize the variations in the
intensities that result from taking images on different chase days. For
every field analyzed, the phase contrast images were juxtaposed to the
corresponding fluorescence images to ensure that any noncell objects or
partially viewed cells were eliminated from the analysis.
Confocal Microscopy--
An axial series of fluorescence images
was obtained with a Zeiss LSM 510 laser-scanning confocal microscope
equipped with a × 63, NA 1.4 plan Apochromat objective (Carl
Zeiss, Inc., Jena, Germany). Red and green images were acquired
sequentially using the 543-nm (exciting the Cy3) and 488-nm (exciting
the fluorescein) lines from a 1.0-milliwatt HeNe laser, and a 25 milliwatt argon laser, respectively. For images acquired in this
manner, cross-talk of FITC fluorescence into the Cy3 channel was not
detectable. 0.5-µm vertical steps were used, with a vertical optical
resolution of less than 1.0 µm.
| |
RESULTS |
Degradation and Release of Internalized 125I-Labeled
fA--
In our previous study, adherent microglia were incubated
with either Cy3-labeled fA42 or 125I-labeled fA42 for
a short period of time and kept in label-free growth medium for up to 3 days. We found that microglia retained Cy3-labeled fA42 for up to 3 days, and only about 30% of internalized 125I-labeled
fA42 was degraded in that time. We wanted to look at the degradation
kinetics of fA for a longer period of time to see if the cells
carried out progressive, slow degradation. We used both fA40 and
fA42, and we observed no difference in the uptake, degradation, or
release between fA40 and fA42. We found that about 60% of
internalized 125I-labeled fA was released after 12 days,
while 40% of the fA still remained cell-associated (Fig.
1A). Only a fraction of the released A was degraded to trichloroacetic acid-soluble fragments. The degradation of fA, as observed by the amount of trichloroacetic acid-soluble material released from microglia, reached 15-20% within
3 days, and then no further degradation was observed (Fig. 1B). However, there was a progressive release of
trichloroacetic acid-precipitable peptide throughout the 12-day chase
time. To ensure that the trichloroacetic acid-soluble material is, in
fact, peptide-associated 125I, we performed a chloroform
extraction of the trichloroacetic acid-soluble fraction of the chase
medium. Over 95% of the radioactivity remained in the aqueous layer,
indicating that there was not a significant amount of free
125I in the trichloroacetic acid-soluble fraction (data not
shown).
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Fig. 1.
Degradation and release of
125I-labeled fA by microglia.
Microglia were incubated for 1 h with 125I-labeled
fA (1 µg/ml), after which the cells were washed and chased for
varying times. In parallel samples, excess Ac-LDL (200 µg/ml) was
added to the radiolabeled fA to determine the extent of nonspecific
uptake, for which all measurements were corrected. At each chase time,
the chase medium was collected and cells were solubilized in 1 M NaOH. The radioactivity in the cells ( ) was measured
(A). The chase medium was subjected to trichloroacetic acid
precipitation for 1 h on ice to monitor degradation. The
radioactivity of trichloroacetic acid-soluble ( ) and trichloroacetic
acid-insoluble ( ) fractions are shown as the percentage of total
counts/min (B). The data presented are averages of the
radioactive counts from three dishes per condition from five different
experiments using fA42. Similar results were obtained using fA40.
Error bars, S.E.
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Population Analysis of Microglia Degrading Cy3-fA--
Since
only a small fraction of internalized fA is degraded by microglia,
it seemed possible that some cells could degrade A much more readily
than others. We wanted to know whether 15-20% of the cell population
is readily degrading fA, or if all the cells are homogeneously
degrading only 15-20% of the internalized fA. We tested these
hypotheses by a quantitative fluorescence microscopy assay using
pulse-chase experiments with Cy3-labeled fA42. The cells were
incubated with Cy3-fA42 for 1 h and chased for various times.
After each chase time, the cells were rinsed, fixed, and observed by
digital fluorescence microscopy. The total fluorescence power in each
cell was quantified to monitor the degradation of Cy3-fA42 as
measured by the decrease in the fluorescence power. After the 1-h
loading pulse, the majority of cells became brightly labeled with
Cy3-fA42 in many small punctate spots scattered throughout the
cytoplasm as reported in a previous study (44). Intensity histograms of
cells over the course of 7 days are shown in Fig.
2. We found that the rate of fA42
degradation is homogenous over the entire population of cells. It
appears that most microglia degrade or release fA slowly.
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Fig. 2.
Loss of Cy3-fA42
from microglia. Microglia were incubated for 1 h with
Cy3-fA42 (1 µg/ml) and chased for various times. After each chase
time, the cells were rinsed extensively, fixed, and imaged by digital
fluorescence microscopy. The Cy3 fluorescence intensity remaining in
each cell over the course of chase was quantified and presented as
histograms. Shown is a representative result from four experiments
conducted on different days.
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The Rate of 2M Degradation in Microglia Is Not
Affected by the Presence of fA--
One possible cause of slow
degradation of cell-associated A would be that the insoluble A
overwhelms the cellular degradative machinery or disrupts the integrity
of lysosomal membranes (57). In order to test this, we preloaded
microglia with unlabeled fA42 and Cy3-labeled fA42 for 3 days.
Cells were observed under the fluorescence microscope to verify that
the cells had accumulated Cy3-fA42 inside the cell (Fig.
3, A and B). This
massive accumulation of fA42 did not affect the viability of the
cell as observed from cell number and morphology. More than 80-90% of
the cells were viable (data not shown), and most of the cells that died detached from the plates and were washed away during rinses before the
solubilization of cells with 1 M NaOH for collection and
radioactivity measurements.
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Fig. 3.
2M degradation by
microglia is not affected by the presence of intracellular
fA42. In 24-well plates, half of the
wells were incubated with unlabeled and Cy3-labeled fA42 and the
other half with no fA42 for 3 days. Phase-contrast (A)
and fluorescence (B) microscopy images of cells incubated
with Cy3-fA42 are shown. Cells were rinsed and chased for 1 h.
Cells were then washed twice with labeling medium and pulsed with
125I-labeled 2M (5 µg/ml) for 45 min. For
both unloaded and preloaded cells, excess unlabeled 2M
(1 mg/ml) was added to some wells along with the radiolabeled
2M as a competitive inhibitor in order to determine
nonspecific uptake. Cell-associated 125I-labeled
2M was measured (C). Cells were washed
extensively and chased for varying times. After each chase time, cells
were solubilized in 1 M NaOH. The radioactivity in the
cells was measured (D). 2M degradation was
assessed by trichloroacetic acid-soluble radioactivity in the chase
medium of fA42 unloaded () and preloaded () samples
(E). Error bars, S.E.
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After rinses with labeling medium, the cells were chased in labeling
medium for 1 h to allow delivery of fA42 into late
endosomes/lysosomes (41). Cells were then washed twice with labeling
medium and pulsed with 125I-labeled 2M (5 µg/ml) for 45 min. 2M enters cells by
receptor-mediated endocytosis and is delivered to late endosomes and
lysosomes, where it is degraded (48). Control cells without the fA42
preload were treated exactly the same way as the preloaded cells prior to the addition of 125I-labeled 2M.
Degradation of 125I-labeled 2M was monitored
by measuring trichloroacetic acid-soluble material released into the
chase medium. We found that there was no difference in the uptake of
2M in microglia with or without fA42 preload (Fig.
3C). In addition, the degradation of 2M was not affected by fA42 preincubation (Fig. 3, D and
E). Over 90% of the total radioactivity from the
internalized 125I-labeled 2M was released
into the medium as trichloroacetic acid-soluble material within 4 h in both fA42-preloaded and unloaded cells (Fig. 3E). In
addition, the rate of degradation of 125I-labeled
2M was not affected by the fA preincubation.
2M Enters the Organelles Preloaded with
fA--
We then verified that the 2M was delivered
to the organelles that contained fA. After short incubations with
fA42 (1-4 h), there was no effect on the degradation of
2M (data not shown), and we know that at these times the
fA42 and the 2M are in the same endosomes and
lysosomes (44). We examined the localization of FITC-labeled
2M after preloading with Cy3-fA42 for 3 days. Co-localization of FITC-2M with Cy3-fA42 was observed
after a 2-h chase. Fig. 4, A
and B, shows a representative single confocal slice of
microglia that have been chased for 2 h. By 6 h of chase, however, all of the FITC-2M was degraded (Fig.
4D), whereas Cy3-fA42 remained in the lysosomes. These
results demonstrate that the endocytosis of 2M and its
intracellular trafficking into degradative organelles are not affected
by fA (in the targeted compartments). Furthermore, the slow
degradation, or the intracellular stability, of fA is not due to a
defect in the lysosomal proteolytic activity. Also, A accumulates in
compartments that still receive incoming material and are
hydrolytically active.
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Fig. 4.
2M is
delivered to fA42 containing organelles.
Microglia plated on coverslip dishes were loaded with 3 µg/ml
Cy3-labeled fA42 for 3 days. Cells were rinsed and pulsed with 5 µg/ml FITC-2M for 45 min. Cells were washed and then
chased for various times. After each chase time, cells were rinsed,
fixed, and imaged by confocal microscopy. Representative cells from a
2-h chase (A and B) and a 6-h chase (C
and D) are shown. Shown are a single confocal fluorescence
optical slice of Cy3-fA42 (A and C) and
FITC-2M (B and D). The
arrows indicate examples of FITC-2M in
compartments previously filled with Cy3-fA42. Scale bar,
10 µm.
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Internalization of 125I-Labeled Soluble A by
Microglia Is Not Blocked by Excess Acetylated Low Density Lipoprotein
or Fucoidan--
It is possible that microglia cannot remove and
degrade fA very well, but they could degrade sA as reported
in vitro in human fibroblasts (58). To examine this
possibility, we tested 1) whether microglia can internalize sA and
2) whether they can degrade internalized sA. Freshly diluted
125I-labeled A was used in similar uptake and
degradation experiments as in Fig. 1. The soluble A preparation was
ultracentrifuged to remove any aggregated A (59-61). Since it has
been shown that A40 is the most abundant A species in
cerebrospinal fluid of AD and control brains (7) and is less
fibrillogenic, 125I-labeled sA40 was initially used to
examine sA uptake and degradation. However, the results of both
sA40 and sA42 were comparable (data not shown). We found that
sA is not internalized by scavenger receptors (Fig.
5). The uptake of
125I-labeled sA40 or sA42 was not blocked by excess
scavenger receptor ligands such as fucoidan and Ac-LDL, which inhibited
the internalization of fA (41).
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Fig. 5.
Internalization of soluble
A is not blocked by scavenger receptor
ligands. 125I-labeled A (4 µg/ml) was diluted in
labeling medium and centrifuged in ultracentrifuge at 100,000 × g for 1 h. Microglia were incubated with the resulting
supernatant alone, with 200 µg/ml Ac-LDL, or with 400 µg/ml
fucoidan for 2 h at 37 °C. The cells were rinsed extensively as
described under "Experimental Procedures." Cells were solubilized
with 1 M NaOH, and the cell-associated radioactive counts
were measured. Both sA40 and sA42 were used. Data presented shows
the average radioactive counts from three dishes per condition from a
representative experiment using 125I-labeled A40.
Error bars, S.E.
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To determine whether there is a concentration dependence of sA
uptake, we incubated microglia with fluorescently (Cy3) and radioactively (125I) labeled sA (1 µg/ml) in
competition with excess unlabeled sA42 in varying concentrations
(Fig. 6). After incubation with the
labeled sA, the cells were washed, and the amount of labeled sA
uptake after internalization was quantified. We found that Cy3-sA42
and 125I-sA40 were taken up nonsaturably (Fig. 6). These
results suggest that sA is internalized by microglia via fluid-phase
pinocytosis, in contrast to receptor-mediated endocytosis of fA
(41). Since this process is not receptor-mediated, we needed to use
higher concentrations of sA and/or longer incubation times to get
measurable uptake.
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Fig. 6.
Nonsaturable uptake of soluble
A. 1 µg/ml Cy3-labeled sA42
(A) or 125I-labeled sA40 (B)
diluted in labeling medium (Dulbecco's modified Eagle's medium with
10 mg/ml bovine serum albumin) was ultracentrifuged at 100,000 × g for 1 h. Microglia were incubated with fluorescently
or radioactively labeled sA alone or with unlabeled sA42 in
varying concentrations for 15 min at 37 °C. For A, cells
were rinsed, fixed, and imaged by digital fluorescence microscopy. The
average fluorescence power per cell was quantified using MetaMorph
imaging software as described under "Experimental Procedures." For
B, cells were rinsed extensively after the pulse and
solubilized with 1 M NaOH, and the radioactivity was
measured. Error bars, S.E.
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Soluble A Is Released from Microglia Mostly as Intact Protein
within 9 h after Internalization--
To analyze the kinetics of
sA degradation, microglia were incubated with 4 µg/ml
125I-labeled sA diluted into protein-free incubation
medium for 2 h at 37 °C, rinsed, and reincubated in the absence
of ligand. As described under "Experimental Procedures," very
little of the A forms insoluble aggregates within the 2-h pulse time
under these conditions. Therefore, the kinetics observed in these
experiments are mainly that of sA. We found that the rate of release
of 125I-labeled sA was quite different from that of
125I-labeled fA. Within 9 h after uptake, about
80% of the 125I-labeled sA was released into the chase
medium with very little intracellular retention (Fig.
7A). In addition, there was
little degradation of 125I-labeled A as determined by
trichloroacetic acid-soluble counts. In fact, within 9 h about
70% of the A was released as intact peptides (Fig. 7B).
Both sA40 and sA42 were used in the degradation study, and again,
the degradation/release kinetics of sA40 were similar to those of
sA42. The release of pinocytosed material is not unusual; many cells
release a significant fraction of internalized solutes back into the
medium (62). However, the time course for such release is often faster
than we have observed for the A. For instance, a large fraction of
insulin is released as intact proteins with a t1/2
of 15 min in fibroblasts (63). The slow release of sA suggests a
more complex itinerary in microglia.
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Fig. 7.
Internalized 125I-labeled soluble
A is released from the cells within 10 h. Microglia were incubated with 125I-labeled sA (4 µg/ml) after ultracentrifugation, as described previously, for 2 h at 37 °C. The cells were rinsed extensively and incubated in chase
medium for various times. Cells were solubilized with 1 M
NaOH, and the cell-associated radioactive counts were measured ( )
(A). The chase medium from each time point was subjected to
trichloroacetic acid precipitation (, trichloroacetic acid-soluble;
, trichloroacetic acid-insoluble) (B). Data presented
show the average of radioactive counts from three dishes per condition
from a representative experiment using 125I-labeled
sA40. Similar results were obtained using 125I-labeled
sA42. Error bars, S.E.
|
|
Soluble A Is Delivered to Late Endosomes and Lysosomes Prior to
Release--
To characterize the compartments containing soluble A
after internalization, microglia were incubated with Cy3-sA42 and FITC-labeled 2M for 1.5 h. We found that
Cy3-sA42 (Fig. 8A) is taken
into the same endosomes as 2M (Fig. 8B). At
1 h of chase time, both Cy3-sA42 (Fig. 8C) and
FITC-2M (Fig. 8D) were concentrated in the
same organelles around the nucleus, consistent with delivery to late
endosomes and lysosomes. This is a strong indication that the
trichloroacetic acid-insoluble A that is released into the medium
passed through late endosomes and lysosomes.
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Fig. 8.
Soluble A is
delivered to late endosomes and lysosomes. Microglia were
incubated with 2 µg/ml Cy3-labeled sA42 (after
ultracentrifugation) and 5 µg/ml FITC-labeled 2M for
1.5 h. Cells were rinsed and chased for 1 h. After chase,
cells were rinsed, fixed, and imaged by confocal microscopy.
Representative cells with no chase (A, Cy3-sA42;
B, FITC-2M) or a 1-h chase (C,
Cy3-sA42; D, FITC-2M) are shown.
Arrows indicate examples of colocalization of the
internalized sA and 2M. Scale bar, 10 µm.
|
|
| |
DISCUSSION |
The mechanisms for the development of amyloid plaque in AD brains
are poorly understood. A is secreted in soluble form by many cell
types throughout the body, and its accumulation in certain regions in
AD brains may involve changes in pathways that lead to production of
fibrillar A. Alternatively, changes in the degradation of soluble
A or fibrillar A could lead to net accumulation of amyloid even
without changes in the rate of amyloid production. Understanding the
mechanisms behind normal clearance of A may provide important
information regarding the pathogenic deposition of A in AD brains.
Our previous studies have shown that microglia can internalize and
degrade fibrillar microaggregates of A (41). These findings have led
us to study further the nature of the interaction between A and microglia.
We found that fA internalized by microglia is partially degraded
over a 3-day period. In this study, we observed about 20% degradation
in 3 days (Fig. 1), which is similar to the degradation we reported in
a previous study over the same time period (44). Analysis of a
population of cells indicated that most cells were capable of limited
degradation of fA (Fig. 2). Surprisingly, there was almost no
further degradation of intracellular fA over a subsequent 12-day
chase. Instead, we observed a slow release of nondegraded fA such
that approximately 35% of the internalized fA was released
undegraded by day 12 of the chase. The mechanism by which microglia
release undegraded A remains to be further investigated. Release of
lysosomal contents has been observed in other cells such as
fibroblasts, and lysosomal release in these cells can be stimulated by
increased intracellular Ca2+ (64).
The fact that there is significant degradation of fA shows that
microglia are capable of degrading fA. It is unclear why the
degradation is only partial and why it stops after a few days. One
possibility is that the hydrolytic properties of the late endosomes and
lysosomes become impaired. There have been reports that internalization
of A may block the normal degradation of proteins in lysosomes and
may disrupt the integrity of lysosomal membranes (57). A related
possibility is that the fA is slowly delivered to a nondegradative,
postlysosomal compartment. We tested these possible explanations for
incomplete degradation by examining lysosomal proteolysis of
internalized proteins by microglia that had taken up massive amounts of
A microaggregates. We found that there was no significant change in
the ability of these fA-engorged cells to internalize or degrade
125I-2M (Fig. 3). Furthermore, when the same
experiment was performed using fluorescently labeled A and
2M, we found that the 2M was delivered to
the same organelles that were previously filled with A (Fig. 4).
After a chase, the fluorescent fA remained in the cells, whereas the
fluorescent 2M was degraded and released. Thus, there is
no general effect of fA on protein degradation in the late endosomes
and lysosomes, and proteolysis can proceed in the organelles that
contain the fA. Since the compartments that have been filled with
fA are hydrolytically active, the resistance to degradation must be
a property of the fA itself. Perhaps the fA is partially
hydrolyzed until only a protease-resistant core is left. Another
possibility is that small amyloid fibrils associate into larger
aggregates in the late endosomes and lysosomes, and these larger
structures present a smaller fraction of the A on the surface, where
they are accessible to proteases. The low pH in the late endosomes and
lysosomes should promote the growth of amyloid fibrils, and the fact
that the compartments containing undegraded fA remain accessible to
newly internalized proteins (Fig. 4B) indicates that the
aggregates could grow intracellularly for days.
Unlike fA, sA is internalized by fluid phase uptake. The uptake
is nonsaturable (Fig. 6), and it is not blocked by ligands for the
scavenger receptors (Fig. 5). In pulse-chase studies using 125I-A, we found that, within 10 h, most of the
sA that entered microglia was released from the cells as nondegraded
peptides, and a small fraction was released as degraded,
trichloroacetic acid-soluble products. The nondegraded A was
released after sA uptake with t1/2 of about
2 h (Fig. 7B).
The fate of molecules that enter cells by fluid phase pinocytosis has
been investigated in several studies. Pinocytosed solutes pass through
early and late endosomes and are delivered to lysosomes. A fraction of
solute molecules can be returned from each of these endosomal/lysosomal
compartments back to the cell surface, and these return pathways may be
similar to those used for the return of peptide-loaded MHC class II to
the plasma membrane (65). In studies of the fate of
[14C]sucrose, a fluid phase marker that is not
metabolized by peritoneal macrophages, it was found that sucrose exited
macrophages by multiple kinetically distinguishable processes (66).
After brief loading pulses, sucrose was released from cells with a
t1/2 of 5 min, but after longer loading pulses it
was released with a t1/2 of 180 min. The rapid exit presumably corresponds to release from early endosomes, and the slow
exit corresponds to release from late endosomes and lysosomes (62). The
time course of release of sA indicates that it is being released
from late endosomes or lysosomes. The transit of sA through late
endosomes was confirmed by co-incubation of Cy3-sA and
FITC-2M, and it was seen that they are internalized into the same endocytic vesicles (Fig. 8). Passage through late endosomes is
somewhat surprising, since it is unclear how the sA avoids degradation in these acidic, hydrolytically active organelles. It is
possible that the sA forms aggregates after internalization. No
aggregated A was detected extracellularly during the pulse with
sA, but it is possible that the soluble peptides aggregate rapidly
inside the cell due to conditions in the intracellular compartments.
The sA could also be protected from proteolysis by associating with
another protein inside the endosomes and lysosomes. Since microglia do
not degrade internalized sA rapidly and since internalized solutes
are delivered to late endosomes and lysosomes, it seems likely that
internalized sA will encounter undigested fA in endosomes and
lysosomes. In that case, sA could become incorporated into existing
amyloid fibrils in acidic endosomes and lysosomes. Progression of
amyloid formation then might involve the incorporation of soluble A
peptides that aggregate with the initially formed seed. In this
scenario, intracellular A fibrils in microglia could serve as
nucleating particles that accumulate soluble A that enters the cell.
In cell-free systems, it has been observed that amyloid formation can
be initiated by "seeding" or "nucleation" with small amounts of
amyloid fibrils (11, 67).
In summary, we have found that microglia degrade only a fraction of the
A that they internalize. Most soluble A is released from
microglia within a few hours, but release of fibrillar A is less
than half complete in 12 days. The fact that microglia do partially
degrade fA suggests that it might be possible to increase this
activity so that microglia could more efficiently protect against the
accumulation of amyloid deposits. On the other hand, the failure to
degrade sA efficiently raises the possibility that once fibrils have
formed, endosomal compartments in microglia could serve as efficient
sites for the growth of amyloid fibrils. Further work will be required
to elucidate the detailed intracellular itineraries of soluble and
fibrillar A and to determine whether the intracellular fates of the
A can be altered to provide more efficient degradation.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Ira Tabas and Cecilia Devlin for
providing Ac-LDL and Lynda Pierini and Lee Cohen-Gould for help with
microscopy and image processing. We are grateful to William Mallet for
a critical reading of the manuscript.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant NS34761.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.
¶
To whom correspondence should be addressed: Weill Medical
College of Cornell University, Dept. of Biochemistry, 1300 York Ave.,
Rm. E215, New York, NY 10021. Tel.: 212-746-6405; Fax: 212-746-8875; E-mail: frmaxfie@mail.med.cornell.edu.
| |
ABBREVIATIONS |
The abbreviations used are:
AD, Alzheimer's
disease;
APP, -amyloid precursor protein;
fA, fibrillar A;
sA, soluble A;
2M, 2-macroglobulin;
FITC, fluorescein isothiocyanate;
LDL, low density lipoprotein.
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TOP
ABSTRACT
INTRODUCTION
