Inheritance of the apoE4 allele (?4) increases the risk of developing Alzheimer’s disease; however, the mechanisms underlying this
a genotype-dependent decrease in apoE levels; ?2/2 ??3/3 ??4/4. Next, we sought to examine the relative contributions of apoE4 and
apoE3 in the ?3/4 mouse brains. ApoE4 represented 30–40% of the total apoE. Moreover, the absolute amount of apoE3 per allele was
and reduced half-life of newly synthesized apoE4 compared with apoE3. Together, these data suggest that astrocytes preferentially
degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Moreover, the genotype-dependent
may directly contribute to the disease progression, perhaps by reducing the capacity of apoE to promote synaptic repair and/or A?
Apolipoprotein E (apoE) is a well characterized 34 kDa protein
Mahley, 1996). ApoE is expressed throughout the brain, and is
produced by astrocytes that secrete apoE as part of a cholesterol-
derived from local synthesis with little contribution from the
periphery (Ferna ´ndez-Miranda et al., 1997). In humans, apoE
allele four of apoE (?4) as a genetic risk factor for Alzheimer’s
disease (AD) has been well established, accounting for between
50–60% of the genetic variation in the disease (Corder et al.,
with a decreased risk for developing AD. ?4 also appears to be a
risk factor for poor outcome after head trauma (Friedman et al.,
1999), cerebral hemorrhage (O’Donnell et al., 2000), and stroke
(Schneider et al., 2005), as well as influencing the age of onset of
other neurodegenerative diseases such as Parkinson’s disease
(Pankratz et al., 2006), multiple sclerosis (Fazekas et al., 2001;
et al., 1996). Although not all of these associations have been
2007; Guerrero et al., 2008). However, together, these data do
suggest that apoE plays a role in the pathophysiology of a wide
range of neurological conditions, not only AD.
Despite knowing for over a decade that apoE4 is a risk factor
for multiple neurodegenerative diseases, the underlying molecu-
lar mechanisms attributing to the risk-factor activity of apoE4
remains unclear. ApoE4 has two major structural characteristics
that distinguish it from apoE3 or apoE2 (Hatters et al., 2006).
First, apoE4 preferentially forms a “molten globule state” that
ultimately reduces the in vitro stability of apoE4 relative to the
other isoforms. Second, apoE4 forms a unique salt bridge inter-
action between Arg-61 in the N terminal and Glu-255 in the
carboxy-terminal domain. This domain interaction results in
apoE4 preferentially binding to lower density lipoprotein parti-
cles and enhanced clearance of apoE4 from the periphery (Raffai
suggest that engineering an apoE4-like, Arg-61 domain interac-
low brain levels of apoE. Here, we confirm and expand on the
findings of Ramaswamy et al. (2005); we demonstrate that astro-
cytes preferentially degrade apoE4, leading to reduced apoE4 se-
TheJournalofNeuroscience,November5,2008 • 28(45):11445–11453 • 11445
cretion and reduced brain apoE levels. Moreover, the genotype-
by ?4 carriers may directly contribute to the disease progression.
Animals. Twelve- to twenty-week-old male ?2/2, ?3/3, ?3/4 and ?4/4
libitum access to food and water. Animal experiments were approved by
the Institutional Animal Care and Use Committee of Wyeth Research.
The evening before the experiment the animals were fasted. The follow-
ing day the mice were anesthetized using isoflurane, plasma and CSF
harvested, saline perfused and the brains removed. CSF was collected as
previously described (DeMattos et al., 2002). Each brain was hemisected
and frontal cortex and/or hippocampus dissected and weighed. Tissues
were homogenized at a ratio of 10 ml/g wet-weight brain in ice-cold
SDS, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, and Complete
protease inhibitors; Roche) using a 10–20 s burst in Polytron homoge-
nizer. Such a detergent-rich lysis buffer has been shown to inhibit apoE
not exposed, may hamper accurate detection of apoE levels by immuno-
assay (Krul and Cole, 1996; Ramaswamy et al., 2005). The homogenates
refrigerated Microfuge. The supernatant was collected and analyzed for
Immunoassays. Human apoE2, apoE3 and apoE4 standards were pur-
chased from Calbiochem. Total apoE and apoE4 in cell culture media or
brain homogenates were measured by a double-sandwich Mesoscale di-
agnostics (MSD)-based immunoassay (Riddell et al., 2007). Briefly, 96-
well streptavidin-coated MSD plates were washed three times in Tris-
buffered saline containing 0.1% Tween 20 (TBST). Ten microliters of
tissue culture supernatant, diluted brain homogenates or human apoE
standards were loaded into wells together with 90 ?l of 1:5000 biotinyl-
ated goat anti-human apoE (Biodesign; K741808) in MSD blocker A.
100 ?l of detection antibody mastermix (1:5000 anti-human pan-apoE
monoclonal antibody (Millipore Bioscience Research Reagents;
For the apoE4 selective immunoassay the anti-human pan-apoE anti-
body was replaced with 1:5000 anti-human apoE4 monoclonal antibody
(MBL International; M067-3). The plates were then washed three times
in TBST and 150 ?l of MSD read buffer-T was added to each well. Plates
were read in a Sector Imager 6000. To ensure that the apoE measured in
three brain homogenates from each genotype were preassayed in tripli-
were determined to be the optimum dilution for brain apoE homoge-
protein assay (Pierce). All apoE measurements were normalized to total
protein in the brain homogenates.
homogenates (10 or 25 ?g/lane) were subjected to SDS-PAGE and then
the goat polyclonal anti-apoE (Biodesign) or apoE4 monoclonal anti-
body (MBL International; M067-3) at concentrations suggested by the
bated with Alexafluor-labeled secondary antibodies, after which the re-
sults were visualized and quantified using the Odyssey infra-red detec-
tion system. Anti-?-tubulin (Upstate) was used as a loading control for
Taqman quantitative RT-PCR. Total RNA was isolated from brain
tissues using TRIzol reagent (Invitrogen) according to the manufactur-
er’s protocol. RNA was purified using RNeasy spin columns (Qiagen),
eluted in RNase-free water and treated with DNase I to remove any
genomic DNA contamination. RNA quality and quantity were assessed
on BioAnalyzer (Agilent Technologies) with RNA 6000 Nano reagents.
Gene-specific mRNA analysis was performed by real-time PCR (Taq-
real-time PCR primers and probes for hApoE and 18 s were purchased
from Applied Biosystems. Amplification was performed using standard-
ized PCR-conditions as recommended by the manufacturer. The stan-
dard curve method was used to estimate specific mRNA concentrations.
PCR results were normalized to 18 s levels.
Astrocytoma cell culture. The human astrocytoma cell lines CCF-
STTG1, U87 and U118 were purchased from ATCC. Cells were main-
tained in DMEM/F12 (3:1) with 10% fetal bovine serum. At 80–90%
confluency, cells were washed with HBSS and then treated with various
reagents in fresh serum-free medium (Neurobasal medium containing
cin with B27 supplementation) for 24 or 48 h. At the end of the treat-
measured using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (Promega).
In some experiments, apoE was detected from media that was concen-
trated 10-fold in Vivaspin concentrators (10,000 molecular weight cut-
g/ml) that were isolated from media by density ultracentrifugation
(LaDu et al., 1998).
ApoE genotyping and DNA sequencing. Genomic DNA was extracted
from cells for PCR restriction fragment length polymorphism (PCR-
RFLP) genotyping, as described previously (Hixson and Vernier, 1990).
Briefly, a 244-bp apoE fragment was amplified by PCR (35 cycles; 97°C
for 1 min, 63°C for 1 min, and 72°C for 1 min) with the primer pair:
5?-GATCAAGCTTCCAATCACAGGCAGGAAG-3? and 5?-GATCCG-
using HhaI and the products visualized on a 20% Tris-buffered EDTA
polyacrylamide gels (Invitrogen). Each genotype gives a specific combi-
nation of HhaI fragment sizes: ?2/2, 91 and 83 bp; ?3/3, 91 and 48 bp;
?4/4, 72 and 48 bp and a mixed genotype: ?2/3, 91, 83, and 48 bp; ?3/4,
91, 72 and 48 bp; ?2/4, 91, 83, 72 and 48 bp (Hixson and Vernier, 1990).
For automatic DNA sequencing of PCR products, the sense primer was
used on the 244-bp product purified from a 1.5% agarose gel.
Primary astrocyte cultures. Mixed glia cultures were prepared using a
protocol adapted from Marriott et al. (1995). Neonatal (postnatal days
itated and cortices were dissected and placed into 4°C HBSS. Cortical
tissue was cut coarsely and incubated in HBSS containing 0.025% tryp-
sin, 0.3% BSA and DNase (40 mg/ml) for 20 min at 37°C. The solution
was replaced by HBSS containing BSA and DNase and triturated 20
times. After allowing for debris to settle the supernatant was passed
through a 40 ?m cell strainer (BD Biosciences) and the process was
repeated twice more. The supernatant was centrifuged at 1000 rpm for 5
min and the pellet was resuspended in 10 ml of DMEM containing 50
units of penicillin, 50 mg/ml streptomycin and 10% heat-inactivated
fetal bovine serum (Invitrogen). Suspended cells were divided into T175
flasks, grown for 10 d in vitro and purified by overnight shaking (120
rpm). Remaining adherent cells, containing ?90% glial fibrillary acidic
protein-positive astrocytes, were plated 48 h before experiments. At 80–
90% confluency, cells were washed with HBSS and incubated in fresh
serum-free medium (neurobasal medium containing 25 mM KCl, 2 mM
glutamine, 100 U/ml penicillin, 100 ?g/ml streptomycin with B27 sup-
plementation) for 24 h. At the end of the incubation, the conditioned
measured using Micro BCA Protein Assay Kit (Pierce). Total cholesterol
gen). Total apoE and apoE4 levels were measured in conditioned media
using the protocol described above.
Pulse-chase experiments and metabolic labeling of cell proteins with
[35S]-methionine. Pulse-chase studies were performed as described pre-
and ?4/4 homozygous mice were cultured for 24 h in serum-free media,
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