Astrocytic Ab1-42 Uptake Is Determined by
Ab-Aggregation State and the Presence of
HENRIETTA M. NIELSEN,1,2,3*SANDRA D. MULDER,1,3JEROEN A. M. BELI€EN,2REN?E J. P. MUSTERS,4
PIET EIKELENBOOM,5AND ROBERT VEERHUIS1,2,3,5
1Department of Clinical Chemistry, VU University Medical Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam,
2Department of Pathology, VU University Medical Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
3The Alzheimer Center, VU University Medical Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam,
4Department of Physiology VU University Medical Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
5Department of Psychiatry, VU University Medical Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
Alzheimer’s disease; amyloid-b clearance; glial cell; apolipo-
Intracerebral accumulation of amyloid-b (Ab) leading to
Ab plaque formation, is the main hallmark of Alzheimer’s
disease and might be caused by defective Ab-clearance.
microglia able to bind and ingest Ab1-42 in vitro, which
appeared to be limited by Ab1-42 fibril formation. We
now confirm that astrocytic Ab-uptake depends on size
and/or composition of Ab-aggregates as astrocytes prefera-
bly take up oligomeric Ab over fibrillar Ab. Upon expo-
(Aboligo) or fibrils (Abfib), a larger (3.7 times more) propor-
tion of astrocytes ingested oligomers compared to fibrils,
as determined by flow cytometry. Ab-internalization was
verified using confocal microscopy and live-cell imaging.
Neither uptake of Aboligo nor Abfib, triggered proinflam-
matory activation of the astrocytes, as judged by quantifi-
cation of interleukin-6 and monocyte-chemoattractant pro-
tein-1 release. Amyloid-associated proteins, including a1-
(SAP), C1q and apolipoproteins E (ApoE) and J (ApoJ)
were earlier found to influence Ab-aggregation. Here,
astrocytic uptake of Abfib increased when added to the
cells in combination with SAP and C1q (SAP/C1q), but
was unchanged in the presence of ApoE, ApoJ and ACT.
Interestingly, ApoJ and ApoE dramatically reduced the
number of Aboligo-positive astrocytes, whereas SAP/C1q
slightly reduced Aboligo uptake. Thus, amyloid-associated
proteins, especially ApoJ and ApoE, can alter Ab-uptake
in vitro and hence may influence Ab clearance and pla-
que formation in vivo.
C 2010 Wiley-Liss, Inc.
Imbalanced production and clearance of amyloid-b
(Ab), resulting in accumulation of Ab in extracellular se-
nile (neuritic) plaques, is considered the main driving
force in Alzheimer’s disease (AD) pathogenesis (Hardy
and Selkoe, 2002). Astrocytes have been proposed to be
important mediators of Ab-clearance (Thal et al., 2000;
Wisniewski and Wegiel, 1991; Yamaguchi et al., 1998)
and several studies with AD transgenic mouse models
have focused on Ab-clearance by animal-derived astro-
cytes (Koistinaho et al., 2004; Pihlaja et al., 2008; Wyss-
Coray et al., 2003). We recently reported that human
astrocytes can internalize Ab1-42 in vitro, and that
increased Ab-aggregate size may limit astrocytic Ab-
uptake. We proposed that astrocytic Ab-uptake might be
determined by the conformation and aggregation state
of Ab (Nielsen et al., 2009).
Ab-aggregate assembly and Ab fibril formation is
probably modulated by factors that are co-localized with
Ab-deposits, amyloid-associated proteins (AAPs), includ-
ing complement components, cytokines and chemokines,
acute-phase proteins, apolipoproteins, and adhesion mol-
ecules (Bauer et al., 1991; Eikelenboom et al., 1994;
Rozemuller et al., 1991; Veerhuis et al., 2005; Zhan
et al., 1995). Several AAPs can form complexes with Ab
and affect Ab fibril formation in vitro (Hughes et al.,
1998; Ma et al., 1994; Veerhuis et al., 2005). Interest-
ingly, many AAPs are found in diffuse plaques at early
stages of plaque formation and might therefore affect
The apolipoprotein E (ApoE) gene is a well-established
susceptibility factor for AD (Avramopoulos, 2009) and
recently, apolipoprotein J (ApoJ) was also identified as a
risk-factor in two AD genome-wide association studies
(Harold et al., 2009; Lambert et al., 2009). Both apolipo-
proteins might be involved in Ab-clearance (DeMattos
et al., 2004). These factors and a1-antichymotrypsin
Grant sponsors: The Swedish Tegger Foundation, Stichting Dioraphte, Interna-
tionale Stichting Alzheimer Onderzoek ISAO; Grant number: 06-517;RV.
*Correspondence to (current address): Henrietta Nielsen; Department of Clini-
cal Sciences, Molecular Memory Research Unit, The Wallenberg Laboratory, 2nd
floor, Sk ˚ ane University Hospital (SUS) Malm€ o, Entrance 46, 205 02 Malm€ o,
Sweden. E-mail: firstname.lastname@example.org
Received 9 February 2010; Accepted 25 March 2010
online5 May2010 in WileyInterScience (www.interscience.
GLIA 58:1235–1246 (2010)
C2010 Wiley-Liss, Inc.
(ACT), a serine protease inhibitor, earlier shown to in-
hibit Ab-degradation in vitro and in vivo (Abraham et
al., 2000), are present in all morphological types of amy-
loid plaques including diffuse (early) as well as senile
(neuritic) plaques. Also, serum-amyloid P component
(SAP) and the complement component C1q co-localize
with amyloid plaques at early stages of development
and appear to affect microglial Ab-clearance in vitro
(Familian et al., 2007; Veerhuis et al., 2003). Most likely,
the development of amyloid plaques depends on a com-
plex interaction between AAPs and Ab itself (Veerhuis
et al., 2005; Zhan et al., 1995), as well as cellular effects
of Ab (Veerhuis et al., 2003). Although many studies
addressed potential effects of AAPs on Ab fibril forma-
tion and glial activation, their effect on cellular Ab-
clearance has remained largely uninvestigated.
Here we investigated uptake of Ab1-42 oligomers
(Aboligo) and Ab1-42 fibrils (Abfib) by cultured human
astrocytes, using confocal microscopy, live-cell imaging,
and flow cytometry. Also, we evaluated the Ab-uptake
modulatory effects of apolipoproteins E and J, SAP, C1q,
and the acute-phase reactant ACT. In addition, since
inflammation is a well-known disease-feature of AD and
mainly driven by glial cells (Eikelenboom et al., 2006),
we in parallel quantified the cellular release of the two
pro-inflammatory agents interleukin-6 (IL-6) and mono-
cyte chemoattractant protein-1 (MCP-1) to evaluate the
possible relation between proinflammatory activation
and Ab1-42 uptake capacity.
MATERIALS AND METHODS
Ab1-42 Preparations With and Without
Ab1-42 (Bachem AG) was dissolved in hexafluoroiso-
propanol (Sigma-Aldrich/Fluka), aliquoted, speed-vac-
uum dried and stored until use, as previously described
labelled) Ab1-42 preparations enriched in oligomers
(Aboligo) and fibrils (Abfib) were prepared essentially as
described before (Chafekar et al., 2008; Dahlgren et al.,
2002). In brief, 100 lg Ab1-42 were dissolved, during
vortexing, in anhydrous dimethyl sulfoxide (DMSO) con-
taining 20 lM FAM-labelled Ab1-42 (FluoAb) (Anaspec
Inc) to obtain a concentration of 2.5 mM Ab. The solu-
tion was sonicated at RT for 10 min and thereafter split
in two equal volumes for preparation of oligomers and
fibrils. For Aboligo, FluoAb was further diluted to a work-
ing solution of 100 lM in phenol-red free Dulbecco’s
modified Eagle’s medium (DMEM) and kept at 4?C for
24 h. For Abfib, FluoAb was diluted to 100 lM in 10 mM
sterile HCl and kept at 37?C for 24 h. Differences in the
Aboligoand Abfibpreparations were recorded using west-
ern blot and electron microscopy (EM). Prior to cell
treatment, the Ab1-42 preparations were diluted 10
times in serum- and phenol red-free culture medium
(treatment medium) and incubated 1 h at RT alone or
mixed with 1 lM and 0.1 lM ACT (Calbiochem), 1 lM
and 0.1 lM ApoE (rPeptide), 1 lM and 0.1 lM ApoJ
(Gift from H. Niessen and N. Hahn at the department of
Pathology of VUmc Amsterdam, The Netherlands; puri-
fied from human plasma by affinity chromatography, as
described (Krijnen et al., 2005)), 85 nM SAP (Calbio-
chem), 5 nM C1q (purified in-house as described before
(Veerhuis et al., 2003)) or SAP/C1q together.
Characterization of Ab Preparations
Aboligoand Abfibpreparations, used for cell treatment,
were size-separated by gel electrophoresis on 10% Tris-
Tricine gels. Proteins were transferred to PVDF mem-
branes (Millipore) using the BioRad Transblot Semi Dry
System. Blots were blocked with PBS containing 5%
milk and 0.05% Tween-20 to avoid non-specific antibody
binding. Ab1-42 was detected using mouse anti-Ab
(6E10; Signet Laboratories) over night at RT followed
by peroxidase conjugated goat anti-mouse Ig (DAKO) for
1 h at RT, and enhanced chemiluminescence (ECL)
Aliquots of the Aboligo and Abfib preparations alone
and selected Ab/AAP mixtures were applied to formvar
carbon-coated copper grids and air-dried at 4?C. Grids
were negatively stained with uranyl acetate for 5 min
and examined with a Zeiss EM109 electron microscope.
The grids were examined at 30.0003 magnification and
electron micrographs show images at a final magnifica-
tion of 90.0003.
Adult primary human astrocytes were isolated from
brain specimens either obtained at autopsy through the
Netherlands Brain Bank or obtained at surgery (focal
cortical temporal resection for intractable complex par-
tial seizures) and cultured as described earlier (De Groot
et al., 1997; Nielsen et al., 2009). Patients gave informed
consent, and the use of the tissue for experiments was
in compliance with the Declaration of Helsinki and
approved by the local Medical Ethics Committee at
VUmc. Primary astrocyte cultures from seven clinically
diagnosed AD patients and five nondemented controls
were included in this study (Table 1). In brief, cerebral
specimens were collected in Dulbecco’s modified Eagle’s
medium (DMEM) and Ham’s F10 Nutrient Mixture
(Ham-F10) 1:1 containing 50 lg/mL gentamycin (Gibco/
Invitrogen). Upon removal of visible blood vessels and
meninges, specimens were minced and digested with
2.5 mg/mL trypsin (Sigma-Aldrich) at 37?C for 20 min in
the presence of bovine pancreatic Dnase I (Boehringer
Mannheim). Cell suspensions were allowed to adhere to
poly-L-lysine (PLL) coated 80 cm2culture flasks (NUNC
NIELSEN ET AL.
A/S) and grown as monolayer cultures in complete cul-
ture medium containing DMEM/HAM-F10 (1:1) supple-
mented with 10% (v/v) fetal bovine serum (FBS) (ICN
Biomedicals), 100IU/mL penicillin and 50 lg/mL strepto-
mycin in humidified air with 5% CO2at 37?C.
For microscopic analyzes with confocal laser scanning mi-
croscopy (CLSM), to determine Ab cellular localization,
astrocytes were cultured using the Lab-Tek? II Chamber
Slide System (4 wells) (Nalge Nunc International) until ?70
to 90% confluent and incubated with 10 lM Aboligoand Abfib
in treatment medium, for 18 h. Following rinsing in PBS,
cells were fixed in 4% formalin at RT for 15 min and then
washed in cold PBS. Before staining, cells were treated
with 1% BSA in PBS containing 0.1% saponine at RT for 30
min. Two cellular targets, the cytoskeleton and the cell
membrane, were stained independently in order to visualize
Ab localization. Cytoskeleton (F-actin) staining was per-
formed with Texas Red-conjugated phalloidin (Invitrogen)
20 min at RT in the dark. To visualize cell membranes, cells
were incubated with rabbit antibodies against human pan-
cadherin (Abcam) at RT for 1h, followed by TRITC-conju-
gated swine-anti rabbit Igs (DAKO) in the dark at RT for 1
h. Cells were then rinsed and cover-slipped using the Vecta-
Shield mounting medium (Vector Laboratories Inc).
CLSM was performed, essentially as described before
(Nielsen et al., 2009; Wouterlood et al., 2007) by use of
a Leica TCS-SP confocal instrument equipped with an
Argon/Krypton gas laser (Leica Microsystems) with 488,
568, and 647 nm excitation wavelength. A Plan-Apochro-
mate Leica 20x/0.7 objective was deployed. The sequen-
tial scanning mode was implemented in all image acqui-
sition with the pinhole fixed at 1.00 arbitrary units
[Airy disk], and a Z-increment fixed at 366 nm. Optical
slices through cells were collected in Z-steps where
recordings alternated between frames, i.e., a section was
scanned first in one channel, then in the next channel.
Channels were carefully configured to detect emission
specifically from one fluorochrome without crosstalk of
the other and to exclude excitation crosstalk of the other
fluorochrome present in the section. Depending on the
thickness of the object between 55 and 78 images of
512 3 512 pixels at 8-bit sampling were recorded corre-
sponding with a stack size between 20 and 29 lm.
Image processing, including 3D-rendering, was per-
formed with the CLSM software, and Amira software.
The relations between Ab, phalloidin-TR labeled F-actin
fibers and pan-cadherin stained cell membranes, were
analyzed in individual optical sections through the cells,
as rotated 3D reconstructions and as merged images.
Representative images from experiments using n 5 4
individual astrocyte cultures, are reported.
To further characterize astrocytic Ab-uptake and cellu-
lar localization of the ingested peptide preparations, we
used live-cell imaging as described before (Ludwig et al.,
2004; Simunek et al., 2005; van der Sar et al., 2003). Astro-
cytes were plated in poly-L-lysine coated optical T-dishes
(Bioptechs Inc) and grown until ?70 to 90% confluent.
Cells were exposed to 10 lM Aboligoand Abfibin treatment
medium for 18 h. After 18 h cells were washed with PBS
and a brightfield differential interference contrast (DIC)
image was taken. In order to stain the glycocalyx of cell
membranes, cells were treated with rhodamine-labeled
wheat germ agglutinin (WGA) (Invitrogen) according to
the instructions of the manufacturer, 30 min prior to imag-
ing. In parallel experiments, cells were incubated with
50 nM Lysotracker (Invitrogen) in order to visualize lyso-
somes. Lysotracker was added 18 h after Ab treatment or
prior to Aboligoand Abfibtreatment, to enable discrimina-
tion between early and late endosomal trafficking. Cells
were imaged in real-time for a maximum of 90 min using
brightfield DIC and fluorescence microscopy using both
green and red darkfield channels at different timepoints.
The image acquisition protocol included 3D stacks of astro-
cyte cell cultures with and without fluorescent Ab proteins.
All live-cell experiments were performed on a ZEISS Axio-
vert 200 Marianas inverted microscope, equipped with a
nanostepper motor (Z-axis increments: 10 nm) and a cooled
CCD camera (Cooke Sensicam, 1280 3 1024 pixels). The
microscope, camera, data viewing and processing were fully
controlled by SlideBookTMsoftware (version 4.2, Intelligent
TABLE 1. Overview of Cases Included in This Study
PMDb(h : min) GenderAge (years) Braak scoreApoE genotype
NA. not applicable.
aClincal diagnosis with or without neuropathological confirmation.
bPMD, post mortem delay time (hours:minutes).
cTissue obtained at surgery.
UPTAKE OF Ab1-42 OLIGOMERS AND FIBRILS
imaging.com]. Representative images from experiments
on individual astrocyte cultures are shown.
Innovations, Denver,CO) [www.intelligent-
Astrocytic binding and uptake of Aboligoand Abfibwas
quantified by flow cytometry as described earlier (Nielsen
et al., 2009). Astrocytes, 5 3 104cells/well, were plated in
24-well plates (Nunc A/S) and cells were allowed to
adhere two days prior to experiments. On the basis of our
previous investigation of Ab-uptake dose-dependency
(Nielsen et al., 2009), cultures were exposed to 10 lM
Aboligoand Abfib, alone and in combination with the differ-
ent AAPs, in treatment medium over night (18 h). Condi-
tioned cell culture medium was collected and stored at
220?C until further analysis for the presence of inflam-
matory molecules monocyte chemoattractant protein-1
(MCP-1) and interleukin-6 (IL-6), and cell viability
(extracellular lactate dehydrogenase (LDH) activity).
Cells were harvested using 0.25% trypsin, centrifuged
5 min at 275g at 4?C, washed in cold FACS buffer (0.25%
BSA in PBS) and resuspended in FACS buffer. Ab-positive
astrocytes, 5,000 counts per condition, were quantified
using the FACS Calibur (BD) with the CellQuest soft-
ware. Cells were gated based on morphological appear-
ance (forward and side scatter). To ensure that Ab-uptake
was quantified using homogenous cell populations (from
individual cultures), the same gates were used for all
experiments. Uptake of Ab was expressed as percentage
of Ab-positive cells. In each individual experiment 1% of
non-Ab treated astrocytes were gated as fluorescence-pos-
itive, to account for astrocytic auto-fluorescence, and used
as reference upon quantification of Ab-positive cells.
Enzyme Linked Immuno-Sorbent Assay
To determine astrocytic proinflammatory activation
and production of inflammatory factors, cell superna-
tants were collected from the astrocyte cultures used for
FACS analyzes and analyzed for MCP-1, using the
MCP-1 DuoSet enzyme linked immuno-sorbent assay
(ELISA) (R&D Systems), and IL-6, assayed with the
Pelipair IL-6 ELISA kit (Sanquin).
To evaluate the cytotoxic effects of the various treat-
ments, we quantified extracellular lactate dehydrogenase
activity (LDH) in the conditioned medium (Korzeniewski
and Callewaert, 1983) collected before cell harvest and
FACS analyzes, as a measure of cell viability. In 96-well
UV-plates (Corning Inc), 50 lL cell-free conditioned
medium were mixed with 25 lL 100 mM potassium phos-
phate buffer (pH 7.4) containing 1.32 mg/mL sodium
pyruvate and 25 lL 1 mg/mL NADH in sterile H2O, and the
rate of decrease in absorbance at 340 nm measured for
10 min at 37?C in a Microplate Spectophotometer SPEC-
TRAmax 250 (Molecular Devices). Conditioned medium
from untreated cells was used as a control (100%) and con-
ditioned medium from cells treated 18 h with 1 lM Stauro-
sporine (Sigma-Aldrich) was used as a positive control for
Statistical analysis was performed using the SPSS soft-
ware (version 16.0 for Windows, SPSS Inc). Differences in
uptake of Aboligoand Abfibbetween the AD and non-AD
cultures were investigated using the independent-samples
t-test and when no statistical differences were found, the
AD and non-AD cultures were pooled to form one group of
independent cultures. Thereafter, uptake of Aboligo and
Abfib, alone and in combination with selected AAPs, and
release of MCP-1, IL-6, and LDH were analyzed using the
paired samples t-test and the Bonferroni correction for
multiple comparisons. Correlations were investigated
using the Pearson correlation test. Results are expressed
as means 6 standard deviation (SD) and, when appropri-
ate, shown as normalized data (percentage of untreated
astrocytes). P < 0.05 was considered significant.
Western Blot and EM-Characterization of
To characterize the molecular content of the Ab1-42
oligomer and fibril- enriched preparations, aliquots from
the freshly made Aboligoand Abfibwere subjected to gel
electrophoresis and western blot (Fig. 1). The western
blot confirmed that we had two different Ab prepara-
tions. The Abfibpreparation mainly contained large Ab
aggregates of >33.1 kDa in size, however, some Ab
monomers and dimers were also present. The Aboligo
preparation also contained some larger Ab aggregates
but was in comparison to Abfibenriched in smaller size
Ab fragments, including monomers, dimers, trimers, tet-
ramers, and larger-sized oligomers (Fig. 1).
The separate characteristics of Aboligoand Abfibprepa-
rations were confirmed upon EM examination at an end
magnification of 90.0003 (Fig. 2A,B). The Aboligoclearly
contained many electron-dense globular, nonfibrillar
structures (Fig. 2A), whereas Abfibexhibited numerous
thread-like fibrillar structures and less globular confor-
mations (Fig. 2B). Next we analyzed the combinations of
Ab and the AAPs (Ab-SAP/C1q, Ab-ApoJ, Ab-ApoE).
Upon co-incubation of Aboligowith 85 nM SAP and 5 nM
C1q, a small increase in number of fibrillar structures
(Fig. 2C) was observed, compared to Aboligoalone (Fig.
2A). In contrast, preparations of Abfib incubated with
SAP/C1q, contained more amorphous aggregates and
less well-defined fibrils (Fig. 2D), than Abfibalone (Fig.
2B). The most dramatic change in appearance was
observed when Aboligo was combined with 1 lM ApoJ
(Fig. 2E). Whereas only small globular electron-dense
NIELSEN ET AL.
Ab species were seen with Aboligoalone (Fig. 2A), Aboligo
in combination with 1 lM ApoJ was found to consist of
larger compact and heavily electron-loaded Ab aggre-
gates (Fig. 2E), possibly representing Ab-ApoJ protein
complexes. These large Ab aggregates were to some
extent also found when 1 lM ApoJ was incubated with
Abfib(Fig. 2F). Only few remnants of the fine meshwork
of fibrillar structures, that could be observed in Abfib
preparations (Fig. 2B), remained upon incubation of
Abfibwith 1 lM ApoJ (Fig. 2F). Compared to ApoJ, co-
incubation with ApoE at 1 lM had only minor effects on
the conformation of Ab. The globular Ab species in Aboligo
were somewhat larger and a slight increase in fibrillar
structures was observed in the presence of ApoE
(Fig. 2G). Abfibin combination with 1 lM ApoE consisted
of a fine fibrillar meshwork, similar to that seen in Abfib
alone (Fig. 2H). However, the fibrillar threads tended to
be less electron-dense than in Abfiband the Abfib/ApoE
preparation also contained a number of globular Ab spe-
cies (Fig. 2H) that were larger than in Abfib(Fig. 2B).
Ab Cellular Localization
By use of CLSM we confirmed cellular Ab-internaliza-
tion and found that smaller sized Ab aggregates were more
easily taken up by the astrocytes. Upon incubation with
the Aboligopreparation, numerous smaller Ab aggregates
were found entangled in the TR-labelled cytoskeleton and
protruding the cells (Fig. 3A). In contrast, only few larger
aggregates were found to be intertwined with the cytoskel-
eton, whereas the majority was bound to the cell surface
when astrocytes were exposed to Abfib(Fig. 3B). To further
investigate cellular uptake of Ab, we also stained the outer
cell membranes of the astrocytes upon exposure to Ab. By
use of the same imaging technique, we could in more detail
visualize and confirm cellular uptake of both Aboligo(Fig.
3C) and, to a lesser extent, Abfib(Fig. 3D). Smaller Ab
aggregates from the oligomeric preparation were nicely
sandwiched between the cell membranes (Fig. 3C) whereas
the larger Ab aggregates were either bound to or embed-
ded in the cell membrane (Fig. 3D).
To confirm the cellular localization of Ab in the living
cell, live-cell imaging was performed on cells exposed to Ab,
for 18 h, and then stained with rhodamine-labeled WGA to
visualize the cellular membranes. Evidently Aboligoco-local-
ized with the cell membranes (Fig. 4C), whereas Abfibdid
not (Fig. 4G). In addition, 3D-reconstructed images demon-
strated the presence of Aboligo (Fig. 4D) but not Abfib
(Fig. 4H) inside the cells. After confirming uptake of Ab
oligomers by living astrocytes, we incubated cells with
LysoTracker? to visualize possible Ab accumulation into
lysosomes. No co-localization of Ab and lysosomes was
found during LysoTracker? treatment, after 18 h of Ab ex-
posure or when LysoTracker? was added to the cells prior
to Ab treatment (data not shown). Clear punctuate staining
of the lysosomes was observed with LysoTracker?, whereas
no such pattern was present for the Ab-preparations (data
not shown). These results indicate that Ab-internalization
does not involve lysosomal processing.
Quantification of Astrocytic Ab1-42 Uptake
Uptake of Aboligoand Abfib
By use of flow cytometry we quantified the fraction of
Ab-positive astrocytes after 18 h exposure to 10 lM of
either Aboligoor Abfib. The fluorescence signal from the
appeared to be higher than when exposed to Abfib, indi-
cating that more Ab was ingested per cell when cells
were treated with Aboligo (Fig. 5A). Interestingly, as
many as 3.7 times more astrocytes were Ab-positive
upon treatment with the oligomeric preparation com-
pared to the fibrillar preparation (P ? 0.001) (Fig. 5B).
Further, we compared the percentage of Ab positive
astrocytes between cultures derived from non-AD and
AD-patients and found a slight, but not statistically sig-
nificant, increase in the AD-derived cultures treated
with Aboligo(53.1% 6 10.7 vs. 74.2% 6 14.6). A trend
towards a higher percentage of Ab positive cells in the
AD group upon treatment with Abfib (8.1% 6 6.7 vs.
19.5% 6 18.1) was observed, however, due to large var-
treatments, after separation using 10% tris-tricine gelelectrophoresis
and detection with anti-Ab (6E10) antibodies. Predominantly high molec-
ular weight Ab-species (>33.1 kDa) were found in the Abfibpreparation
but also some Ab monomers (?4 kDa) and dimers (?8kDa). Less high mo-
lecular weight Ab-species were found in the Aboligopreparation, whereas
more smaller Ab-species (<33.1 kDa) were seen, mainly consisting of
monomers, dimers, trimers (?12 kDa) and tetramers (?16 kDa).
Western blot of aliquots from Abfiband Aboligoused for cell
UPTAKE OF Ab1-42 OLIGOMERS AND FIBRILS
iance in Abfib-uptake between cells from different cases,
the difference was also not statistically significant.
There was no difference in uptake of either Ab prepara-
tion between cultures from ApoE4 carriers vs. noncar-
riers (Aboligo, 69.5% 6 17.9 vs. 60.7% 6 17.6 and Abfib,
18.6% 6 19.6 vs. 10.8% 6 11.0).
Influence of AAPs
A summary of the AAPs included in the present study
and their effect on Ab uptake is presented in Fig. 5C.
Because of differences in cell yield between isolations, not
all conditions could be applied to all cultures. The number
H) individually or in combination with AAPs that significantly altered astrocytic Ab-uptake. (A, B) Ab
alone, (C, D) SAP/C1q 1 Ab, (E, F) 1 lM ApoJ 1 Ab, (G, H) 1 lM ApoE 1 Ab.
Electron micrographs (magnified 390.000) of 10 lM Aboligo(A, C, E, G) and Abfib(B, D, F,
NIELSEN ET AL.
of experiments carried out at each condition is presented
in Fig. 5C. In line with our earlier findings (Nielsen et al.,
2009), ACT, tested at 0.1 lM and 1 lM, did not signifi-
cantly influence Ab uptake, neither of Aboligonor of Abfib.,
although the fraction of Ab positive astrocytes slightly
increased upon treatment with Abfiband both concentra-
tions of ACT (Fig. 5C). Complement component C1q, at a
concentration of 5 nM, and also SAP (85 nM) alone, had no
impact on uptake of both preparations of Ab (Fig. 5B).
However, C1q (5 nM) in combination with 85 nM SAP
(SAP/C1q) increased uptake of Abfibby 48.6% (P 5 0.034),
whereas Aboligouptake was reduced by 7.8% (P 5 0.036).
Also, we investigated the influence of ApoJ and ApoE at
two different concentrations, 0.1 lM and 1 lM (Fig. 5C).
ApoJ at 1 lM dramatically reduced (88.9%; P 5 0.001) the
fraction of Ab positive cells upon co-incubation with Aboligo,
whereas a slight, but nonsignificant increase in uptake of
Abfibwas seen. At a 10-fold lower concentration (0.1 lM),
ApoJ reduced the uptake of Abfibby 24.1% (P 5 0.042) but
had no effect on Aboligouptake (Fig. 5C). Similar to ApoJ,
ApoE at 1 lM strongly reduced (79.9%; P 5 0.038) the per-
centage of Ab positive astrocytes, when cells were treated
with Aboligo. This effect of 1 lM ApoE was not observed
when cells were treated with Abfib. Whereas ApoJ already
had an effect on Abfibuptake at a 0.1 lM concentration, no
significant effect of 0.1 lM ApoE was observed, despite a
similar trend of reduced numbers of Ab positive astrocytes
after treatment with both Aboligoand Abfibthat had been
preincubated with ApoE (Fig. 5C).
Astrocytic Proinflammatory Response and
Levels of the pro-inflammatory factors MCP-1 and IL-6
are increased in AD brain tissue extracts and both MCP-1
and IL-6 were localized in neurons as well as astrocytes
(Sokolova et al., 2009). A study on rat astrocytes sug-
gested differential effects of oligomeric and fibrillar Ab1-
42 on astrocyte-mediated inflammation (White et al.,
2005). Recently, we found that astrocytic release of MCP-
1 decreased upon exposure to 10 lM fluorescent Ab1-42
preparations (green) and TR-phalloidin (F-actin cytoskeleton) (red,
images A,B) or anti-PAN cadherin (cell membrane) (red, images C–F).
Analyzes were performed by optical Z-stacking through the cells (20–29
lm stacks) and 3D-reconstruction of the obtained Z-stacks. Side-view of
astrocyte monolayer after exposure to (A) Aboligo, reveals various Ab-
fragments intermingled with (arrow heads) and protruding through the
F-actin cytoskeleton structures; (B) Abfib, shows few Ab-aggregates
entangled with the cytoskeleton (arrow heads), most Ab is predomi-
nantly located on top of the cell. In a slightly tilted overview of the cell
layer, (C) a large part of the Aboligopreparation appears to be present
CLSM of astrocytes after 18 h exposure to FAM-labeled Ab-
on top of the cells, bound to the cell surface (filled arrow head); how-
ever, a substantial amount of Ab is partly embedded by or located
underneath the cell membrane (open arrow heads). On the other hand,
(D) the Abfib-preparation forms massive aggregates that are mainly
attached to the cell surface (arrow heads). In a tilted side-view, E)
Aboligofragments are neatly sandwiched in-between the cell membranes
whereas Ab-aggregates from the (F) Abfib-preparation appear larger
and mostly stuck to or embedded in the cell membranes facing the
lumen (up). Scale bars represent 10 lm. [Color figure can be viewed in
the online issue, which is available at www.interscience.wiley.com.]
UPTAKE OF Ab1-42 OLIGOMERS AND FIBRILS
preparations (Nielsen et al., 2009). We now investigated
the release of cellular MCP-1 and also of the proinflam-
matory cytokine IL-6 by astrocytes upon treatment with
Aboligand Abfibusing similar experimental conditions as
in the previous study (Nielsen et al., 2009).
Release of MCP-1 at baseline (untreated cells) was
similar between astrocytes derived from AD (n 5 7) and
non-AD (n 5 3) (863.3 6 760.5 pg/mL versus 1079.0 6
932.1 pg/mL, P 5 0.709) patients and between ApoE4 car-
riers (n 5 6) noncarriers (n 5 3) (902.1 6 806.0 versus
1011.7 6 1005.7 pg/mL, P 5 0.863). Therefore, the MCP-1
data were pooled. Release of MCP-1 was significantly
reduced upon treatment with either 10 lM Aboligoor Abfib,
as compared to baseline (Fig. 6A). The astrocytes secreted
62.8 6 19.3% of the MCP-1 levels released at baseline,
after exposure to Abfib(P 5 0.023, n 5 11), and 73.1 6
29.8% of baseline levels after treatment with Aboligo(P 5
0.019, n 5 11) (Fig. 6A). However, we found no difference
in MCP-1 release from cells treated with Abfib versus
Aboligo and also no link between MCP-1 levels and
Ab-uptake. Further, the addition of the different AAPs
did not have any major effects on the MCP-1 release nei-
ther when comparing with baseline secretion or release
after treatment with the two different Ab preparations
alone (data not shown). Of note however was the slight
increase in MCP-1 release after treatment with Abfiband
Aboligoin combination with 1 lM ACT (Fig. 6A).
In line with the results on MCP-1 release at baseline,
there was no difference in IL-6 secretion between astro-
cytes derived from AD (n 5 7) and non-AD patients (n 5 3)
(25.9 6 25.2 pg/mL versus 29.5 6 17.6 pg/mL, P 5 0.826)
and astrocytes from ApoE4 carriers (n 5 6) and noncar-
riers (n 5 3) (28.9 6 26.1 pg/mL 629.9 6 pg/mL, P 5
0.954). Therefore, the IL-6 results from individual astro-
cyte cultures were pooled. Unlike MCP-1, IL-6 secretion
from astrocytes treated to 10 lM Aboligodid not differ from
untreated astrocytes, whereas it was slightly increased
when compared with cells exposed to 10 lM Abfib(P 5
0.018) (Fig. 6B). Co-incubation of the astrocytes with
Aboligoor Abfibtogether with the AAPs had no statistically
significant impact on IL-6 release, although co-treatment
of Abwith either 1 lM ACTor 1 lM ApoJ appeared to have
a promoting effect on IL-6 release (Fig. 6B).
Interestingly however, we observed a negative correlation
between IL-6 release and the fraction of Ab positive astro-
cytes upon exposure to either Aboligo(r 5 20.682, P 5 0.021,
n 5 11) or Abfib(r 5 20.660, P 5 0.027, n 5 11) alone.
Several studies have shown Ab1-42 oligomers to be
more toxic to neurons than Ab1-42 fibrils (Dahlgren et al.,
2002; Resende et al., 2008). We analyzed the release of
intracellular LDH, as a read-out for cytotoxicity, in our
Aboligoand Abfibtreated astrocyte cultures, and used cells
trast images of astrocytes after 18 h exposure to FAM-labelled (green)
Aboligo (A–D) and Abfib (E–H). Cell membranes were visualized by a
wheat germ agglutinin (WGA) staining (red) (B-D, F-H). Cells were
imaged in real-time. In Aboligotreated astrocytes, Aboligo, and WGA were
found highly colocalized (yellow) (C), indicating that the oligomeric Ab is
Live cell imaging and brightfield differential interference con-
taken up by the cells whereas little or no colocalization was found
between fibrillar Ab and WGA (G). This is emphasized by the 3D recon-
struction-images with encircled cell-outlines, in which Aboligo-WGA (D)
but not Abfib-WGA (H) colocalization is clearly visible inside the cell.
[Color figure can be viewed in the online issue, which is available at
NIELSEN ET AL.
exposed to 1 lM staurosporine for 18 h as a positive con-
trol. Whereas our positive control induced a 6.6 6 3.4
(n 5 3) fold increase in LDH release (data not shown), the
LDH release from the Ab-treated cultures was similar to
baseline levels (Fig. 6C) and was not linked to the per-
centage of Ab-positive cells. Of note, an over-all trend of
slightly elevated LDH levels in cultures treated with
Aboligoversus Abfibwas observed. However, we found no
significant increase in LDH release induced by either
Aboligoor Abfibalone or in combination with the AAPs,
compared to untreated control cells (Fig. 6C).
In this study we show that primary human astrocytes
internalize smaller size Ab1-42 oligomers more avidly
than fibrillar Ab1-42. Not only a larger fraction of the
astrocytes became Ab-positive upon exposure to the
Aboligo preparation, compared with Abfib, but also the
fluorescence intensity per cell was higher, suggesting
that the amount of ingested Ab is larger in the Aboligo-
treated population (Fig. 5A,B). Cytokine IL-6 release by
whereas MCP-1 release was significantly decreased,
compared to untreated astrocytes. In contrast, oligo-
meric Ab1-42 was earlier shown to induce a profound
early inflammatory response in cultured rat astrocytes,
as judged by IL-1b release, whilst fibrillar Ab1-42
appeared to foster a more chronic inflammation (White
et al., 2005). On the basis of our current and earlier
observations (Nielsen et al., 2009), we believe that
human astrocytes react differently to Ab than rodent
astrocytes. Our data on MCP-1 release suggest that Ab
alone has an anti-inflammatory effect on human astro-
cytes. The preferred uptake of Aboligoover Abfib, without
inflammatory response, might be of significant relevance
for the understanding of plaque development, the role of
sentative histogram displaying background fluorescence of untreated
astrocytes and the distribution of Ab-positive cells when exposed to
FAM-labelled Aboligoand Abfib(10 lM), for 18 h. B: Percentage of Ab-
positive astrocytes (n 5 12 independent astrocyte cultures) upon incu-
bation with 10 lM Aboligo and Abfib. C: Ab-positive astrocytes after
treatment with Aboligo and Abfib in combination with several AAPs,
expressed as % of Ab-positive astrocytes upon exposure to Aboligoand
Abfibalone (solid line 5 100%). Results from individual astrocyte cul-
tures (a minimum of three cultures per condition), are presented as
means and standard deviations (whiskers). [Color figure can be viewed
in the online issue, which is available at www.interscience.wiley.com.]
Astrocytic Ab-uptake, as determined using FACS. A: Repre-
LDH (C) as indicators of proinflammatory activation and cytotoxic
effects of Ab-treatments (Ab 6 AAPs). Cell-responses from treated cells
were compared to baseline untreated cells (solid line 5 100%). Results
from a minimum of n 5 3 individual astrocyte cultures are presented
(same as Fig. 3) as means and standard deviations (whiskers).
Assessment of astrocytic release of MCP-1 (A), IL-6 (B),
UPTAKE OF Ab1-42 OLIGOMERS AND FIBRILS
astrocytes as Ab-scavengers and their role in AD associ-
ated neurodegenerative and neuroregenerative proc-
Deposition of Ab and formation of nonfibrillar diffuse
plaques are the earliest signs of Ab plaque formation
(Griffin et al., 1995; Ikeda et al., 1990; Rozemuller et al.,
1989). However, diffuse plaques can also be found in
nondemented subjects and their prevalence does not cor-
relate with cognitive impairment (Delaere et al., 1991).
Several unknown disease-causing and modifying factors
may lead to formation of senile plaques, consisting of
fibrillar Ab, and engagement of activated microglia and
Whether these plaque types evolve through a series of
events from diffuse plaques or develop through distinct
pathways, is not clear. Aggregation of Ab and plaque
formation may be the net result of various factors,
including local concentrations of (various forms of) Ab
and of a number of AAPs. Interestingly, human micro-
glia and astrocytes seem to respond to Ab, in association
with AAPs, differently. For example, cultured primary
response to a mixture of Ab, SAP and C1q (Veerhuis et
al., 2003), whereas astrocytes exposed to the same mix-
ture (however prepared with pre-aggregated Ab), as
shown here, do not.
The pre-clinical phase of AD is believed to be long. Kok
and colleagues recently reported AD-related lesions in
nondemented individuals already at 30 years of age (Kok
et al., 2009). At what stage the presumably benign and
possibly natural Ab-deposition starts taking a malign
pathway towards pathological Ab-accumulation and se-
nile/neuritic plaque formation, is unknown. Interestingly,
diffuse plaque Ab-granules were found within astrocytes
leading to the speculation that astrocytes phagocytize Ab
and prevent plaque formation (Yamaguchi et al., 1998).
Hence, hampered astrocytic Ab-clearance may precede
and/or be responsible for accumulation of Ab (Wyss-Coray
et al., 2003) with subsequent increase in Ab plaque load
and clinical onset of AD.
To date, mechanisms underlying astrocytic Ab-uptake
are not known. Potential Ab-receptors, the low-density
lipoprotein receptor (LDLR) and the LDLR-related pro-
tein-1 (LRP-1), were identified on astrocytes in vivo and
in vitro. However, expression of both was unaltered
upon Ab-exposure and receptor-blockage did not inhibit
astrocytic Ab-uptake (Wilhelmus et al., 2007). In sup-
port, we found the phagocytosis inhibitor cytochalasin B,
proven to inhibit Ab-phagocytosis by cultured microglia
(Familian et al., 2007), unable to inhibit astrocytic
Ab-uptake (Nielsen et al., 2009). Together, the lack of
Ab—lysosome co-localization during live-cell imaging
and our inability to block or track Ab-uptake, suggests
that other than receptor-mediated mechanisms direct
astrocytic Ab-uptake. As Ab40 can be ingested by neu-
rons via a nonendocytotic, nonsaturable and energy in-
dependent process, i.e. passive diffusion (Kandimalla
et al., 2009) and microglia can internalize soluble Ab1-
42 through a nonsaturable macropinocytic mechanism
in vitro and in vivo (Mandrekar et al., 2009), an inter-
esting speculation, which needs further investigation,
would be that similar nonendocytotic mechanisms may
also apply to Ab-uptake by astrocytes.
About forty AAPs have been identified (Akiyama
et al., 2000). The AAPs C1q and SAP co-localize with dif-
fuse and fibrillar Ab plaques, and seem to be a prerequi-
site for microglia clustering and activation. In vitro
Ab-SAP/C1q mixtures activate human microglia to a
larger extent than Ab alone (Veerhuis ea 2003) and Ab-
uptake by microglia is reduced in the presence of SAP/
C1q (Familian et al., 2007), but until now, studies on the
effect of SAP/C1q on astrocytic Ab-uptake have been
lacking. We found SAP/C1q, combined with Aboligo, to
significantly decrease the fraction of Ab-positive astro-
cytes, whereas SAP/C1q added to Abfib increased the
percentage of Ab-positive cells. Thus, SAP/C1q affects
astrocytic uptake of Ab oligomers and fibrils in opposite
directions. Neither Ab-SAP/C1q nor SAP or C1q alone,
induced astrocytic IL-6 and MCP-1 release. Reports on
effects of SAP on Ab are conflicting. SAP alone was
shown to inhibit Ab fibril formation (Janciauskiene
et al., 1995) but also to stabilize already formed Ab fibrils,
thereby preventing Ab proteolysis (Tennent et al., 1995).
Low cerebrospinal fluid SAP concentrations were associ-
ated with a two-fold increased risk of progression to AD in
individuals with mild cognitive impairment (Verwey
et al., 2008). Whether this increased risk is due to insuffi-
cient availability of SAP to scavenge Ab or to promoting
effects on plaque-formation, is still unknown. Further, Ab
in combination with another AAP, ACT, can modify the
global gene-expression profile of human astrocytes in cul-
ture (Baker et al., 2007) and lead to rat astrocyte activa-
tion (Hu and Van Eldik, 1999). We indeed observed
increased IL-6 and MCP-1 release from Ab/ACT treated
astrocytes, however were unable to statistically confirm
these differences. Earlier findings, that ACT inhibits
Ab-clearance (Abraham et al., 2000), could not be
confirmed in our recent study on human astrocytic
Ab-uptake in vitro (Nielsen et al., 2009). Here, we con-
firmed and extended those results as ACT did not appear
to have any effect on astrocytic uptake of either oligomeric
or fibrillar Ab in vitro. Thus, whereas ACT has little or no
effect on astrocytic Ab-uptake, it might induce proinflam-
Apolipoprotein E, as well as ApoJ, have earlier been
attributed involvement in Ab-clearance (DeMattos et al.,
2004). In two recent, genome-wide association studies of
AD, as expected ApoE, and in addition ApoJ (CLU) and
complement (3b/4b) receptor 1 (CR1), were found to be
associated with AD (Harold et al., 2009; Lambert et al.,
2009). Decreased ApoE levels in brain of ApoE4-carriers
might contribute to AD progression, possibly by hamper-
ing Ab-clearance (Riddell et al., 2008). We found no dif-
ference in Ab-uptake between astrocytes from ApoE4
carriers and noncarriers. Interestingly, endogenous pro-
duction and release of ApoE, by cultured human astro-
cytes, is very low and does not differ between cultures of
astrocytes derived from ApoE41 and ApoE42 donors
(Bruinsma et al., 2010). We therefore believe that our
results are unbiased in regard of endogenously produced
NIELSEN ET AL.
ApoE by the astrocytes. In an AD mouse-model, ApoE
promoted astrocyte colocalization and degradation of de-
posited Ab peptides (Koistinaho et al., 2004) and, based
on human fetal astrocyte study results, a role for ApoJ
in Ab-degradation was proposed (Nuutinen et al., 2007).
We now found that ApoE and ApoJ dramatically reduced
the Aboligouptake by adult human astrocytes, as addi-
tion of ApoJ and ApoE to the Aboligopreparation reduced
the fraction of Ab-positive astrocytes by nearly 90% and
80% respectively (Fig. 3B). ApoE and ApoJ had limited
or no effect on Abfibuptake. In support, Bruinsma et al.
found that ApoE significantly reduced the accumulation
at the cell surface of cultured human astrocytes and
internalization of the mutant AbGlu22Gln1-40 (Dutch
type) peptide (Bruinsma et al., 2010). These and our
findings contrast current knowledge on the effects of
ApoE and ApoJ on Ab-clearance. ApoE was reported to
be essential for astrocytic Ab-degradation from mouse
brain sections as astrocytes from ApoE 2/2 mice could
not degrade or internalize Ab from the tissue slices
(Koistinaho et al., 2004). Also, ApoE and ApoJ coopera-
tively suppressed cerebral Ab-levels and Ab-deposition
in an AD transgenic mouse model (DeMattos et al.,
2004). Further, ApoE enhanced endolytic Ab-degrada-
tion by cultured microglia and promoted extracellular
Ab-proteolysis by the insulin-degrading enzyme (Fan
et al., 2009; Jiang et al., 2008). The discrepancies
between these results and those from our study might
be explained by species-dependent differences, but also
by the involvement of Ab-clearance mechanisms other
than those moderated by astrocytes, for instance micro-
glial phagocytosis and transport across the blood-brain-
Many of the AAPs included in this study can bind to
Ab and form complexes, and affect Ab-aggregation
(Fraser et al., 1993; Ma et al., 1994; Oda et al., 1995;
Veerhuis et al., 2003). Here, especially ApoJ seemed to
alter the conformation of the two different Ab-prepara-
tions. Although ApoE exerted only modest effects on
Aboligo, as judged by EM, the effect on astrocytic Aboligo-
uptake was large. Whether our observed differences
upon combination of Ab with several AAPs are related
to different mechanistic routes for uptake of oligomeric
and fibrillar Ab or involvement of alternate receptors for
AAP-Ab-complexes, is hard to say and needs more inves-
In conclusion, cultured primary human astrocytes
preferably take up Ab1-42 oligomers over fibrils, without
evidence of pro-inflammatory activation. Apolipoproteins
E and J, can significantly alter uptake of oligomeric Ab
and hence may influence Ab-clearance and deposition at
different stages of plaque formation. Our results, in com-
bination with the results from previous in vivo and
in vitro, immunohistochemical and genome-wide associa-
tion studies, strongly warrant further investigation to
elucidate the roles of ApoE and ApoJ in AD pathogene-
sis. To extend current knowledge on cellular Ab-clear-
ance and to serve as a potential future pharmacological
target, astrocytic Ab-internalization mechanisms and Ab
interactions with AAPs, need to be further investigated.
The authors wish to acknowledge the Netherlands Brain
Bank. They thank Marlies Jacobs, Harry Twaalfhoven,
Jan Fritz, Rien Dekker, and Sylvia Bogaards for excellent
technical assistance. They also acknowledge Prof. Hans
Niessen and Nynke Hahn of the Cardiovascular research
group at Pathology, VUmc for the kind gift of ApoJ.
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