Expression Of Human Apolipoprotein E3 Or E4 In The Brains Of Apoe ...

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Expression of Human Apolipoprotein E3 or E4 in the Brains of
Apoe2/2 Mice: Isoform-Specific Effects on Neurodegeneration
Manuel Buttini,1,4 Matthias Orth,1,2 Stefano Bellosta,1,2 Hassibullah Akeefe,1 Robert E. Pitas,1,2,5
Tony Wyss-Coray,1,4 Lennart Mucke,1,4 and Robert W. Mahley1,2,3,5
1Gladstone Institute of Neurological Disease, 2Cardiovascular Research Institute, and Departments of 3Medicine,
4Neurology, and 5Pathology, University of California, San Francisco, California 94141-9100
Apolipoprotein (apo) E isoforms are key determinants of susceptibility to Alzheimer’s disease. The apoE4 isoform is the
major known genetic risk factor for this disease and is also
associated with poor outcome after acute head trauma or
stroke. To test the hypothesis that apoE3, but not apoE4,
protects against age-related and excitotoxin-induced neurodegeneration, we analyzed apoE knockout (Apoe2/2) mice expressing similar levels of human apoE3 or apoE4 in the brain
under control of the neuron-specific enolase promoter. Neuronal apoE expression was widespread in the brains of these
mice. Kainic acid-challenged wild-type or Apoe2/2 mice had a
significant loss of synaptophysin-positive presynaptic terminals
and microtubule-associated protein 2-positive neuronal dendrites in the neocortex and hippocampus, and a disruption of
neurofilament-positive axons in the hippocampus. Expression
of apoE3, but not of apoE4, protected against this excitotoxininduced neuronal damage. ApoE3, but not apoE4, also protected against the age-dependent neurodegeneration seen in
Apoe2/2 mice. These differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of
Alzheimer’s disease and to the poor outcome after head trauma
and stroke associated with apoE4 in humans.
Key words: apolipoprotein E; Alzheimer’s disease; apoE
transgenic mice; excitotoxicity; apoE knockout mice; neurodegeneration
Apolipoprotein (apo) E is a 34 kDa protein that participates in
the transport of plasma lipids and in the redistribution of lipids
among cells (Mahley, 1988). Of the three common apoE isoforms
in humans (Utermann et al., 1977), apoE4 is a major risk factor
for Alzheimer’s disease (AD) (Corder et al., 1993; Strittmatter et
al., 1993; Mayeux et al., 1995; Farrer et al., 1997) and for poor
outcome after acute head injury (Nicoll et al., 1996; Teasdale et
al., 1997) or stroke (Slooter et al., 1997). The most common
isoform, apoE3, differs from apoE4 by only a single amino acid
(Weisgraber et al., 1981; Rall et al., 1982).
A role for apoE in neural injury and repair processes was
established well before this molecule was implicated in AD
(Elshourbagy et al., 1985; Ignatius et al., 1986; Pitas et al., 1987;
Mahley, 1988). More recent studies in Apoe2/2 mice further
suggested that apoE helps protect the brain against acute injury
(Chen et al., 1997) and maintain neuronal integrity during aging
(Masliah et al., 1995). Other studies have not detected age-related
neurological abnormalities in Apoe2/2 mice (Anderson et al.,
1998; Fagan et al., 1998).
The first suggestion that apoE was involved in AD came from
the immunohistochemical localization of apoE in two hallmark
lesions of AD brains: amyloid plaques and neurofibrillary tangles
(Namba et al., 1991). Additional studies suggested that apoE4
may contribute to these lesions through pathogenic interactions
with the Ab peptide of amyloid plaques (Wisniewski and Frangione, 1992; Ma et al., 1994; Sanan et al., 1994; Strittmatter et al.,
1994) and the tau protein of neurofibrillary tangles (Strittmatter
et al., 1994).
Another mechanism by which apoE might be involved in neurodegenerative processes is by isoform-specific effects on neurite
extension and cytoskeletal stability (Mahley, 1988; Mahley et al.,
1995; Weisgraber and Mahley, 1996). Addition of apoE3 to neuronal cultures stimulates neurite outgrowth, stabilizes microtubules, and is associated with an accumulation of cytoplasmic
apoE, whereas apoE4 does not have these effects (Nathan et al.,
1994, 1995; Bellosta et al., 1995b; Ji et al., 1998).
The above observations raise the possibility that repair and
remodeling of neurons in response to injury proceed more effectively in the presence of apoE3 than apoE4, and that this is a
reason why apoE4 acts as a susceptibility factor for age-related
neurodegenerative diseases such as AD. To test this apoE injury/
repair hypothesis in vivo, we expressed apoE3 and apoE4 at
comparable levels in the CNS of Apoe2/2 mice using the neuronspecific enolase (NSE) promoter and studied the isoform-specific
effects on neurodegeneration associated with aging and excitotoxicity in these mice. The rationale for neuronal targeting was based
on a number of observations. Apolipoprotein E immunoreactivity
in neurons was reported in human AD brains (Han et al., 1994;
Bao et al., 1996; Metzger et al., 1996) and in rat brain after
ischemia (Horsburgh and Nicoll, 1996). Neuronal expression of
apoE mRNA was detected in human brain (Xu et al., 1999).
These data indicate that apoE could exert critical effects within
neurons. NSE-driven expression of human apoE results in the
Received Dec. 29, 1998; revised March 19, 1999; accepted March 29, 1999.
This research was funded in part by a Cambridge NeuroScience/Gladstone
collaborative research agreement. M.O. was supported in part by the Deutsche
Forschungsgemeinschaft. We thank Ricky Quan and Carol Lin for excellent technical support; Sylvia Richmond for manuscript preparation; Gary Howard and
Stephen Ordway for editorial assistance; and John C. W. Carroll, Stephen Gonzales,
and Chris Goodfellow for graphics and photography.
Drs. Buttini and Orth contributed equally to this study.
Correspondence should be addressed to Dr. Robert W. Mahley, Gladstone Institute of Neurological Disease, P.O. Box 419100, San Francisco, CA 94141-9100.
Dr. Bellosta’s current address: Institute of Pharmacological Science, University of
Milan, Via Balzaretti 9, 20133 Milan, Italy.
Copyright © 1999 Society for Neuroscience 0270-6474/99/194867-14$05.00/0
The Journal of Neuroscience, June 15, 1999, 19(12):4867–4880
secretion of human apoE isoforms into the culture medium of
transfected neuronal cells (Bellosta et al., 1995b). Also, human
apoE isoforms exert similar effects on cultured neuronal cells,
whether they are added to the culture medium in purified form
(Nathan et al., 1994), expressed in neuronal cells via stable
transfection (Bellosta et al., 1995b), or secreted from cocultured
astrocytes (Sun et al., 1998). Our comparison of NSE-apoE3 and
NSE-apoE4 mice revealed that apoE3 protects the CNS against
excitotoxin-induced and age-related neurodegeneration, whereas
apoE4 does not.
Animals. One hundred seventy-eight 3- to 9-month-old mice, weighing
25–35 gm, were studied. Mice were kept under a 12 hr light /dark cycle
with free access to sterile water and food (PicoLab Rodent Diet 20,
#5053, PMI Nutrition International, St. Louis, MO). Four genotypes
were analyzed: wild-type mice, Apoe 2/2 mice, NSE-apoE3 mice, and
NSE-apoE4 mice. NSE-apoE transgenic mice on the Apoe 2/2 background were generated as follows. NSE-apoE transgenes were injected
individually into one-cell embryos (ICR strain) by standard procedures;
transgenic lines were established from transgenic founders. NSE-apoE3
and NSE-apoE4 lines with matching cerebral levels of transgene expression were selected and crossed with Apoe 2/2 mice (Piedrahita et al.,
1992) provided by Dr. Nobuyo Maeda (University of North Carolina,
Chapel Hill, NC). After elimination of wild-type Apoe alleles in two
generations of breedings among the resulting offspring, transgenic mice
were crossed with Apoe 2/2 mice (C57BL/6J-Apoe tm1Unc) from Jackson
Laboratories (Bar Harbor, ME) to generate NSE-apoE3 and NSEapoE4 mice that were at least 75% C57BL/6J. Crosses of NSE-apoE3 or
NSE-apoE4 with C57BL/6J-Apoe tm1Unc mice from Jackson Laboratories
also yielded nontransgenic Apoe 2/2 littermates (n 5 27). Comparison of
the latter mice with age-matched C57BL/6J-Apoe tm1Unc mice from Jackson Laboratories (n 5 23) revealed no significant differences in any of the
variables examined (data not shown). Therefore, these two cohorts of
mice were combined (Apoe 2/2 mice) in our statistical analyses.
Genotyping of transgenic mice. Mice transgenic for NSE-apoE3 or
NSE-apoE4 were identified by Southern blot analysis of genomic tail
DNA using a DNA probe for human APOE (Bellosta et al., 1995a).
NSE-apoE3 and NSE-apoE4 mice were differentiated by PCR. Because
the human APOE intron 3 was included in the NSE-apoE4 but not in the
NSE-apoE3 construct, the amplicon generated with intron 3-spanning
primers (forward primer: nucleotides 3158–3175; reverse primer: nucleotides 3815–3834, GenBank accession number M10065) was 670 base
pairs (bp) in NSE-apoE4 mice and 100 bp in NSE-apoE3 mice. Proteinase K-digested tail tissue (1:100 dilution, 2 ml) was subjected to touchdown PCR (Hecker and Roux, 1996) in a total reaction volume of 25 ml
with each primer (0.2 mM), dNTPs (dATP, dCTP, dGTP, dTTP, 200 mM
each), and 0.15 ml of AmpliTaq GoldR DNA polymerase (Perkin-Elmer,
Norwalk, CT). The reaction was run on a GeneAmp PCR System 9600
thermocycler (Perkin-Elmer). PCR products were analyzed on 1.5%
agarose gels. To determine the Apoe knockout status of the mice, total
plasma cholesterol levels were measured with a cholesterol measurement
kit (Sigma, St. Louis, MO). Analysis of brain mRNA by RNase protection assay (RPA) (see below) confirmed the absence of mouse apoE
mRNA in all mice with cholesterol levels .30 mg/dl (data not shown).
Kainic acid injections. Kainic acid crosses the blood–brain barrier and
induces excitotoxic CNS injury, particularly in the hippocampus and
neocortex (Strain and Tasker, 1991; Masliah et al., 1997). Kainic acid
(Sigma) was dissolved in saline (0.9%) and injected intraperitoneally at
18 or 25 mg/kg body weight in one dose. Within ;15 min, all mice
developed seizures. Seizure activity was assessed as described (Schauwecker and Steward, 1997). There were no differences in kainic acidinduced seizures across groups of mice with respect to overall incidence,
time period between injection and seizure onset, intensity, or duration of
seizures (data not shown). This suggests that brain penetration of kainic
acid was similar in all the mice. Control animals were injected with saline
and did not develop seizures. Mice were killed 6 d after the injection of
kainic acid or saline.
Tissue preparation. Mice were anesthetized with chloral hydrate and
flush-perfused transcardially with 0.9% saline. Brains were removed and
divided sagittally. One hemibrain was post-fixed in phosphate-buffered
4% paraformaldehyde, pH 7.4, at 4°C for 48 hr for vibratome sectioning;
the other was snap frozen and stored at 270°C for RNA or protein
analysis. Postmortem brain tissues from humans with or without AD
were obtained from Dr. Eliezer Masliah (University of California, San
Diego, CA) and from Dr. Tom M. Hyde (National Institute of Mental
Health, Bethesda, MD).
RNA extraction and analysis. Total RNA was isolated from tissues with
TRI-Reagent (Molecular Research Center, Cincinnati, OH) or Tripure
(Boehringer Mannheim, Indianapolis, IN). RNA was analyzed by solution hybridization RPA with antisense riboprobes complementary to
human apoE mRNA [nucleotides 281–469 of APOE cDNA (GenBank
accession number M12529)] or b-actin mRNA [nucleotides 480–559 of
mouse b-actin cDNA (GenBank accession number M18194)]. Because
the apoE riboprobe also protects a smaller fragment of endogenous
mouse apoE mRNA sequence, both human and mouse apoE mRNAs
could be identified with this probe. The RPAs were performed essentially
as described (Bordonaro et al., 1994). Briefly, sample RNA (10 mg)
hybridized to 32P-labeled antisense riboprobes was digested with 300
U/ml RNase T1 (Life Technologies, Gaithersburg, MD) and 0.5 mg/ml
RNase A (Sigma) in 100 ml of digestion buffer, followed by protein
digestion with 10 mg/ml proteinase K (Sigma). RNA was isolated with 4
M guanidine thiocyanate and precipitated in isopropanol. Samples were
separated on 5–6% acrylamide/8 M urea Tris-borate EDTA gels, and the
dried gels were exposed to XAR or Biomax MS film (Kodak, Rochester,
NY). Levels of specific transcripts were estimated by quantitating probespecific signals with a phosphorimager (FUJI-BasIII, Fuji, Tokyo, Japan); b-actin signals were used to correct for differences in RNA content /
loading (Johnson et al., 1995).
Analysis of CSF. After methoxyflurane overdose and exsanguination by
cardiac puncture, CSF was obtained from nine NSE-apoE3, nine NSEapoE4, and eight Apoe 2/2 mice. We modified the procedure described by
Carp et al. (1971) by using a 25 gauge needle attached to silicon tubing
(0.012 inch internal diameter) and piercing the dura mater tangentially.
Slight negative pressure was exerted with a tuberculin syringe to start the
flow. From each adult mouse, ;10 ml of CSF was obtained from the
cisterna magna with little or no contaminating blood. The CSF was
centrifuged in a desktop centrifuge to remove contaminating cells, kept
at 4°C, and used within 3 d for Western blotting and quantitation of
apoE. Equal volumes of CSF from the different cohorts of mice were
loaded on the gels.
Western blot analysis. Brain homogenates from hemibrains were prepared with a triple detergent lysis buffer (Sambrook et al., 1989) and
protease inhibitors [phenylmethylsulfonyl fluoride (100 mg/ml), aprotinin
(1 mg/ml), and complete inhibitor (23, catalog no. 1836145, Boehringer
Mannheim)]. Insoluble material was removed by centrifugation. The
protein concentration in the supernatant was determined with a modified
Bradford method (Pierce, Rockford, IL), and sample protein concentrations were equalized with lysis buffer. SDS loading buffer was added, and
the samples were heated to 95°C for 5 min. To quantitate apoE in brain
tissue and CSF, samples and purified apoE standards (provided by Dr.
Karl Weisgraber, Gladstone Institute of Neurological Disease) were
separated by SDS-PAGE, electrotransferred to nitrocellulose membranes
(Bio-Rad, Hercules, CA), and blocked with PBS containing 5% dried
milk and 0.05% Tween. The blots were incubated in polyclonal goat
anti-human apoE antibody (1:1000; Calbiochem, San Diego, CA) or in
polyclonal rabbit anti-mouse apoE antibody (1:1000; provided by Dr. Jan
Borén, Gladstone Institute of Cardiovascular Disease). The bound primary antibodies were detected with horseradish peroxidase-conjugated
species-specific antibodies (Amersham, Arlington, IL). Immunodetection was performed with SuperSignal Ultra (Pierce) or ECL (Amersham)
according to the manufacturer’s instructions, and the blots were exposed
to x-ray film (Biomax MR, Kodak). For semiquantitative assessments of
apoE, known quantities of purified human plasma apoE3 or apoE4
[prepared as described by Rall et al. (1986) and provided by Dr. Karl
Weisgraber] or recombinant mouse apoE (provided by Dr. Li-Ming
Dong, Gladstone Institute of Cardiovascular Disease) were run as standards on the same gels. For quantitation, exposures of Western blots with
densities within the linear range of the film were scanned, and the density
of the bands was determined by inflection point analysis with Advanced
Quantifier software (BioImage, Ann Arbor, MI).
Immunohistochemistry. Post-fixed tissues were cut into 40-mm-thick
sections with a vibratome and incubated in 0.3% H2O2 in PBS for 20 min
to quench endogenous peroxidase activity. To facilitate penetration of
antibodies, sections used for immunoperoxidase staining were preincubated for 4 min in 1 mg/ml proteinase K in a buffer containing 250 mM
NaCl, 25 mM EDTA, 50 mM Tris/HCl, pH 8. To block nonspecific
reactions, all sections were incubated for 1 hr in 15% normal donkey
4868 J. Neurosci., June 15, 1999, 19(12):4867–4880 Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology
serum (Jackson ImmunoResearch, West Grove, PA) in PBS or for 7 min
in Superblock (Scytec, Logan, UT), followed by a 1 hr incubation in PBS
with the primary antibody: polyclonal goat anti-human apoE (Calbiochem) diluted 1:4000 (immunofluorescent staining) or 1:10,000 (immunoperoxidase staining) to detect human apoE, or polyclonal rabbit antirat apoE diluted 1:1000 (gift from Dr. Karl Weisgraber) to detect murine
apoE. Sections were then washed twice in PBS and incubated for 1 hr
with the secondary antibody: fluorescein isothiocyanate (FITC)–(Jackson ImmunoResearch) or biotin-coupled (Vector, Burlingame, CA) antigoat to detect antigen-bound anti-human apoE or FITC-coupled antirabbit (Vector) to detect antigen-bound anti-rat apoE. After three
washes in PBS, immunofluorescently labeled sections were mounted in
VectaShield (Vector) and viewed with a MRC-1024 laser scanning confocal system (Bio-Rad) mounted on an Optiphot-2 microscope (Nikon,
Tokyo, Japan). For immunoperoxidase staining, secondary antibody
binding was detected with the ABC-Elite kit (Vector).
The intensity of human apoE immunolabeling of neurons in brains of
NSE-apoE mice was determined on immunofluorescently labeled sections with the MRC-1024 system and Lasersharp (Bio-Rad) software. A
10 mm line was drawn through the cytoplasm of five randomly selected
neocortical neurons per animal. The intensity of the pixels across this
line was determined, and the mean pixel intensity per line was calculated.
Double-labeling for human apoE and cell-specific markers was performed essentially as described above except that sections from transgenic animals were incubated with anti-microtubule-associated protein 2
(MAP-2) antibody (1:40 dilution; Boehringer Mannheim) together with
anti-human apoE, and sections from wild-type animals were incubated
with anti-glial fibrillary acidic protein (GFAP) antibody (1:500 dilution,
Boehringer Mannheim) together with anti-rat apoE. To detect primary
antibody binding, sections were incubated for 1 hr in a mixture of
secondary antibodies (1:100 dilution; Jackson ImmunoResearch): an
FITC-conjugated donkey anti-goat (to detect anti-human apoE), an
FITC-conjugated donkey anti-rabbit (to detect anti-rat apoE), and a
Cy5-conjugated donkey anti-mouse (to detect anti-MAP-2 or antiGFAP). After three 10 min washes in PBS, sections were mounted under
glass coverslips with VectaShield (Vector) and viewed by confocal microscopy as described above. The Cy5 and FITC channels were viewed
individually, and the resulting images were pseudocolored in red (Cy5)
or green (FITC) with Adobe Photoshop (version 4.0, Adobe Systems,
San Jose, CA). Omission of primary antibodies or incubation of sections
with mismatched primary and secondary antibodies resulted in no signal.
To exclude the possibility that the signals collected in the FITC channel
originated from emission light from the Cy5 fluorophore and vice versa,
sections labeled with FITC-conjugated secondary antibodies were imaged in the Cy5 channel, and sections labeled with Cy5-conjugated
secondary antibodies were imaged in the FITC channel. No signals were
detected under these control conditions.
Semiquantitative assessment of neurodegenerative changes. Brain sections immunolabeled for MAP-2 (a marker of neuronal cell bodies and
dendrites) or synaptophysin (a marker of presynaptic terminals) were
analyzed semiquantitatively with a Bio-Rad MRC-1024 laser scanning
confocal microscope, mounted on a Nikon Optiphot-2 microscope and
running Lasersharp software, essentially as described (Masliah et al.,
1992; Toggas et al., 1994). Neuronal integrity was assessed in the neocortex and in the stratum radiatum of the hippocampus (CA1 subfield) in
four sections per animal (two for each marker). Binding of primary
antibodies (Boehringer Mannheim) was detected with an FITC-labeled
secondary antibody (Vector). Sections were assigned code numbers to
ensure objective assessment, and codes were not broken until analysis
was complete. For each mouse, we obtained four to eight confocal images
(three to four per section) of the neocortex and two to four confocal
images (one to two per section) of the hippocampal CA1 subfield, each
covering an area of 210 3 140 mm. The iris and gain levels were adjusted
to obtain images with a pixel intensity within a linear range. Four scans
were averaged (Kalman filter) to obtain each final image. Each final
image was processed sequentially in Lasersharp with the following edgeenhancement filters: C7a (for images of MAP-2-labeled sections); C9a,
C3b, and C7a (for images of the neocortex on synaptophysin-labeled
sections); and C9a, C7a, C3b, and C9a (for images of the hippocampus
on synaptophysin-labeled sections). Digitized images were transferred
to a Macintosh computer and analyzed with NIH Image. The area
of the neuropil occupied by MAP-2-immunolabeled dendrites or by
synaptophysin-immunolabeled presynaptic terminals was quantified and
expressed as a percentage of the total image area as described (Masliah
et al., 1992). This approach for the semiquantitative assessment of
neurodegeneration has been validated in various experimental models of
neurodegeneration (Toggas et al., 1994; Masliah et al., 1995) and in
diseased human brains (Masliah et al., 1992).
To assess further the integrity of neuronal structures, brain sections
were immunolabeled with an antibody against phosphorylated neurofilaments in neuronal axons (1:3000 diluted SM31; Sternberger Monoclonals, Lutherville, MD). Antigen-bound antibody was detected with an
FITC-labeled anti-mouse secondary antibody (Vector), diluted 1:75, and
imaged with a laser scanning confocal microscope.
ELISA measurement of synaptophysin in neocortical tissue. Neocortical
tissue from each hemibrain was homogenized with a Kontes motorized
pestle (Fisher Scientific, Pittsburgh, PA) in 0.8 ml ice-cold homogenization buffer (1.85 mM NaH2PO4 , 8.4 mM Na2HPO4 , 150 mM NaCl, 5 mM
benzamidine, 3 mM EDTA, 1 mM MgSO4 , 0.05% sodium azide, pH 8),
and sonicated for 30–60 sec. Homogenates were centrifuged at 2400 3
g for 10 min at 4°C. The supernatant was then ultracentrifuged
(100,000 3 g for 1 hr at 4°C). The resulting pellet (particulate fraction)
was resuspended in 300–400 ml of homogenization buffer, and the
protein concentration was determined by a Bradford assay (Bio-Rad) per
manufacturer’s instructions.
For ELISA measurements of synaptophysin, wells of tissue culture
plates (Costar, Corning, NY) were coated for 14–16 hr at 4°C with
neocortical particulate fractions (0.5 mg of protein). Nonspecific binding
sites were blocked with 2% donkey serum (Jackson) in PBS for 30 min at
room temperature. The anti-synaptophysin antibody (Dako, Carpinteria,
CA), diluted 1:5000 in PBS with 0.5% donkey serum, was applied for 90
min at room temperature. After three 10 min washes with PBS containing 0.05% Tween 20, plates were incubated for 90 min at room temperature with horseradish peroxidase-conjugated anti-rabbit antibody (Amersham) diluted 1:4000 in PBS. After another three 10 min washes with
PBS containing 0.05% Tween 20, the reaction was developed with
o-phenylenediamine dihydrochloride peroxidase substrate tablets (Sigma) per manufacturer’s instructions. The reaction was stopped after 15
min by adding 25% H2SO4 , and the absorbance was measured at 492 nm
with an ELISA reader. Absorbance values for wells in which the incubation with anti-synaptophysin antibody was omitted were subtracted
from the values obtained. Triplicate absorbance values were obtained for
the neocortical particulate fraction of each animal and averaged. In
preliminary experiments, the linear range of the ELISA was 0.1–1 mg of
protein from neocortical particulate fractions (data not shown). Examination of the particulate fraction of mouse liver, an organ lacking
synaptophysin by Western blotting, showed no absorbance (data not
shown). This result confirmed the specificity of the ELISA.
Statistical analyses. Quantitative data are expressed as mean 6 SEM.
Differences between means were assessed by unpaired two-tailed Student’s t test. Differences among means were evaluated by one-way
ANOVA followed by Dunnett’s or Tukey-Kramer post hoc test. The null
hypothesis was rejected at the 0.05 level.
Generation of transgenic mice
The rat NSE promoter directs pan-neuronal expression of fusion
gene constructs in the CNS of transgenic mice (Forss-Petter et al.,
1990). Neuro-2a cells stably transfected with minigenes encoding
human apoE3 or human apoE4 placed downstream of the NSE
promoter secrete human apoE into the cell culture medium
(Bellosta et al., 1995b). These NSE-apoE3 and NSE-apoE4 transgenes (Fig. 1) were used to generate mice expressing apoE3 or
apoE4 in the brain. Nine NSE-apoE3 and 12 NSE-apoE4 transgenic founder mice were identified by Southern blot analysis. Two
lines of transgenic mice that showed comparable levels and distribution of apoE3 versus apoE4 in the brain were selected and
crossed onto the Apoe knockout (Apoe2/2) background as described in Materials and Methods.
Four groups of mice were analyzed in detail: wild-type mice,
Apoe2/2 mice, and Apoe2/2 mice heterozygous for the NSEapoE3 transgene (NSE-apoE3 mice) or the NSE-apoE4 transgene
(NSE-apoE4 mice).
Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology J. Neurosci., June 15, 1999, 19(12):4867–4880 4869
Tissue-specific distribution of apoE3 and apoE4
Human apoE expression in NSE-apoE3 and NSE-apoE4 mice,
determined by RPA, was found primarily in neural tissues and
gonads (Fig. 2A), as observed previously for NSE-driven expression of an indicator gene (Forss-Petter et al., 1990). Immunoblotting showed no human apoE in the plasma of NSE-apoE mice,
and plasma lipoprotein cholesterol levels in the NSE-apoE mice
were similar to those in nontransgenic Apoe2/2 littermate controls (data not shown). Plasma apoE is derived almost exclusively
from the liver, with little, if any, contribution from the CNS
(Linton et al., 1991).
ApoE mRNA and protein levels in the brain and CSF
NSE-apoE3 and NSE-apoE4 mice showed similar steady-state
levels of human apoE mRNA in their brains (Fig. 2B,C). These
levels were similar to those found in human frontal cortex (Fig.
2C). Human apoE protein levels, assessed by Western blot analysis, were also similar in human and transgenic mouse brains (Fig.
3A); no apoE was detected in Apoe2/2 mice (data not shown).
Although apoE is produced exclusively by neurons in the brains
of NSE-apoE mice, in the brains of humans apoE is produced by
neurons and astrocytes (Xu et al., 1999). Because the precise
proportion of apoE produced by neurons and glia in the human
brain remains unknown, the levels of apoE in the brains of
NSE-apoE mice and humans may not be directly comparable.
Densitometric scanning of gels confirmed that apoE levels in
NSE-apoE3 and NSE-apoE4 mice were similar (Fig. 3B). A
tendency toward higher levels of apoE in NSE-apoE4 mice was
not statistically significant ( p 5 0.79). Both mouse and human
brain apoE appeared to be highly sialylated, because they showed
two major bands in the 34–38 kDa range (Fig. 3A). This finding
is consistent with previous results (Pitas et al., 1987). The human
apoE3 and apoE4 in the transgenic mouse brains were intact,
because no significant degradation products were found.
Apolipoprotein E-containing lipoproteins in the CSF originate
in the CNS (Linton et al., 1991). Western blot analysis demonstrated similar levels of human apoE in CSF from NSE-apoE3
Figure 1. NSE-apoE transgenes. The rat NSE promoter (Forss-Petter et
al., 1990) was used to direct the expression of two distinct human apoE
minigenes in neurons (Bellosta et al., 1995a). From 59 to 39, the apoE3
minigene consists of part of the untranslated exon 1 (from the AvrII site),
the first intron, and the first 6 bp of exon 2 from the human APOE gene,
a fragment of human apoE cDNA contributing the entire apoE coding
region, and a genomic segment representing exon 4 noncoding sequence
and 112 bp of 39 untranslated region including the polyadenylation signal.
The apoE4 minigene was similar in structure but also included the third
intron of the human APOE gene. The sequences of the coding regions of
both transgenes were compared to ensure that the only difference between
them was the base change in exon 4 encoding cysteine ( C) in apoE3 and
arginine (R) in apoE4 at amino acid position 112. 1, 2, 3, and 4 indicate
exons of the human APOE gene.
Figure 2. Expression of NSE-apoE transgenes at the RNA level. Total
RNA was extracted from different tissues, and steady-state apoE mRNA
levels were determined by RPA with an apoE antisense riboprobe that
allows differentiation of human from mouse apoE transcripts. A, Representative autoradiograph revealing similar expression patterns of human
apoE mRNA across different organs and CNS regions in NSE-apoE3 (top
panel ) and NSE-apoE4 (bottom panel ) mice. Note the predominant
expression in CNS, eyes, and gonads. The signal in the kidney lane in the
bottom panel is an artifact caused by incomplete RNase digestion of the
sample. B, Comparison of cerebral apoE mRNA levels in NSE-apoE mice
and controls (n 5 2/group). RNA was extracted from entire hemibrains of
7- to 9-month-old NSE-apoE3 (lanes 1, 2), NSE-apoE4 (lanes 3, 4 ),
wild-type (lanes 5, 6 ), and Apoe 2/2 (lanes 7, 8) mice and analyzed by
RPA. The lef tmost lane shows signals of undigested ( U ) radiolabeled
probes; the other lanes contained the same riboprobes plus either tRNA
(D; no specific hybridization) or brain RNA samples, digested with
RNases. As outlined in Materials and Methods, the apoE riboprobe
protects a larger fragment of human apoE mRNA and a smaller fragment
of mouse apoE mRNA (protected mRNAs are indicated on the right).
Note the similar levels of human apoE mRNA expression in brains of
NSE-apoE3 and NSE-apoE4 mice. Comparable results were obtained in
additional cohorts of 7- to 9-month-old mice (n 5 9) and in corresponding
groups of 3- to 4-month-old mice (n 5 6) (data not shown). C, Semiquantitative comparison of human apoE mRNA levels in brain tissues of
NSE-apoE mice and humans. Signals from RPAs on total RNA extracted
from entire hemibrains of NSE-apoE3 (n 5 4) or NSE-apoE4 mice (n 5
6) or from the midfrontal gyrus of humans without dementia (n 5 9) were
quantitated by phosphorimager analysis essentially as described (Rockenstein et al., 1995). No statistically significant differences were identified
when the three groups were compared by ANOVA or when the two
groups of transgenic mice were compared by unpaired, two-tailed Student’s t test.
4870 J. Neurosci., June 15, 1999, 19(12):4867–4880 Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology
and NSE-apoE4 mice (Fig. 3A). No apoE was detected in the
CSF of Apoe2/2 mice (data not shown).
Immunohistochemical localization of apoE3 and apoE4
in brains of NSE-apoE mice
To exclude potentially confounding regional differences in transgene expression, we used an antibody against human apoE to map
human apoE expression in immunolabeled brain sections from
NSE-apoE3 and NSE-apoE4 mice. Brains from both groups
showed similar widespread neuronal expression of human apoE,
which was most prominent in the neocortex and hippocampus
(Fig. 4). This pattern is consistent with that of other NSE-driven
transgenes (Forss-Petter et al., 1990; Mucke et al., 1994).
Confocal microscopy of immunolabeled brain sections from
NSE-apoE mice and from a human AD case revealed similar
intraneuronal distributions of apoE3 and apoE4 in the transgenic
mice and confirmed the presence of human apoE in neurons of
the AD brain (Fig. 5). In transgenic brains, apoE3 and apoE4
were identified in a patchy distribution throughout most of the
neuronal soma with clear sparing of the nucleus (Fig. 5A,B,D,E);
little human apoE was detected in neuronal axons or dendrites
and none in non-neuronal cells (data not shown). In the human
AD case (APOE e3/e4), intraneuronal apoE immunoreactivity
was somewhat more diffuse and extended into neuronal processes
(Fig. 5H,I). Double labeling with antibodies against human apoE
and the neuronal marker MAP-2 confirmed the neuronal identity
of the brain cells expressing human apoE in NSE-apoE3 and
NSE-apoE4 mice (Fig. 6).
Comparison of the neuronal human apoE immunofluorescence
signals in 3- to 4- and 7- to 9-month-old NSE-apoE3 and NSEapoE4 mice (n 5 4–5/group) showed no significant differences in
immunostaining intensity (data not shown). These results are
consistent with those obtained by RPA (Fig. 2B,C) and Western
blot analysis (Fig. 3).
Differential effects of apoE3 and apoE4 on excitotoxininduced neurodegeneration in Apoe2/2 mice
Excessive stimulation of glutamate receptors by excitatory amino
acids, such as glutamic or kainic acid, results in neuronal damage
(excitotoxicity) and is one of the main mechanisms of neuronal
injury in neurodegenerative diseases (Meldrum and Garthwaite,
1990; Lipton and Rosenberg, 1994). To test whether there is an
apoE isoform-specific effect on excitotoxin-induced neurodegeneration, we injected NSE-apoE3 and NSE-apoE4 mice (both on
the Apoe2/2 background) with kainic acid. Control mice were
injected with saline. Kainic acid- and saline-injected Apoe2/2 and
wild-type mice served as additional controls.
Inspection of hematoxylin/eosin-stained sections revealed no
obvious neuronal loss in the hippocampus or neocortex of any of
the kainic acid-injected groups of mice (data not shown), consistent with a previous study reporting that the C57BL/6J strain is
resistant to excitotoxin-induced loss of neuronal cell bodies
(Schauwecker and Steward, 1997).
To detect more subtle types of neurodegeneration, neocortical
and hippocampal sections were immunolabeled for synaptophysin, MAP-2, or phosphorylated neurofilaments and imaged by
confocal microscopy. Systemic injection of kainic acid has previously been shown to induce a significant loss of MAP-2- and
synaptophysin-immunoreactive neuronal structures in mice on
the C57BL/6J background (Masliah et al., 1997). The percentage
area of neuropil occupied by immunolabeled presynaptic terminals or neurites was determined by computer-aided analysis of
confocal images as described in Materials and Methods.
We found that apoE3 effectively protected against excitotoxininduced neurodegeneration, whereas apoE4 did not (Fig. 7, E,F vs
G,H; Fig. 9, C vs D). NSE-apoE3 mice showed no significant loss
of synaptophysin-positive presynaptic terminals in the neocortex
(Figs. 7E, 8A) after injection of 18 or 25 mg/kg kainic acid.
However, a significant loss of MAP-2-positive neuronal dendrites
in the neocortex of these mice was observed after injection of 25
mg/kg kainic acid (Fig. 8B). NSE-apoE3 mice showed no disruption of neurofilament-positive hippocampal axons after injection
of 18 mg/kg kainic acid (Fig. 9C). In contrast, NSE-apoE4 mice
showed significant loss of neocortical synaptophysin-positive presynaptic terminals (Figs. 7G, 8A) and MAP-2-positive neuronal
dendrites (Figs. 7H, 8B) after injection of 18 or 25 mg/kg kainic
acid. NSE-apoE4 mice also showed severe disruption of
neurofilament-positive hippocampal axons after injection of 18
mg/kg kainic acid (Fig. 9D). Apoe2/2 and wild-type mice also
showed a significant loss of neocortical synaptophysin-positive
presynaptic terminals (Figs. 7A,C, 8A) and MAP-2-positive neuronal dendrites (Figs. 7B,D, 8B) with either dose of kainic acid, as
well as disruption of hippocampal axons after injection of 18
mg/kg kainic acid (Fig. 9A,B). Similar genotype effects on
synaptophysin-positive presynaptic terminals and MAP-2positive neuronal dendrites were observed in the hippocampus
(data not shown).
Figure 3. Human apoE3 and apoE4 in brains and CSF of transgenic
mice. A, Western blot analysis showing apoE expression in whole-brain
homogenates (n 5 2 mice/genotype, 50 mg protein/ lane) and CSF (14
ml / lane) from NSE-apoE3 and NSE-apoE4 mice. Human brain (occipital
lobe) homogenate (50 mg protein/ lane) and human apoE3 (5 ng/ lane)
standards are shown as controls. B, The apoE contents of brains and CSF
were estimated by densitometric scanning of gels and by using human
apoE standards. The apoE content in brains of transgenic mice was 4.4 6
0.4 mg apoE/gm protein for NSE-apoE3 mice (n 5 9), and 4.6 6 0.5 mg
apoE/gm protein for NSE-apoE4 mice (n 5 13). The apoE content in the
CSF of mice from both genotypes was 2.8 ng/ml (pooled from 6
Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology J. Neurosci., June 15, 1999, 19(12):4867–4880 4871
Figure 4. Neuronal expression of human apoE in NSE-apoE3 and NSE-apoE4 mouse brain. Immunoperoxidase staining for human apoE revealed
widespread neuronal labeling in different brain regions of NSE-apoE3 and NSE-apoE4 mice. No apoE labeling was seen in Apoe 2/2 control mice. Note
the similar apoE expression pattern in NSE-apoE3 and NSE-apoE4 mice. Note also that human apoE immunoreactivity is present in neuropil as well
as in neuronal cell bodies in the transgenic mice. Scale bars: first row of panels, 400 mm; all other panels, 200 mm.
4872 J. Neurosci., June 15, 1999, 19(12):4867–4880 Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology
Differential effects of apoE3 and apoE4 on age-related
neurodegeneration in Apoe2/2 mice
To determine whether there is an age-related loss of neuronal
structures in Apoe2/2 mice, as has been found by some (Masliah
et al., 1995) but not by others (Anderson et al., 1998), we analyzed
neuronal integrity in Apoe2/2 mice at 3–4 and 7–9 months of age.
Compared with age-matched wild-type controls, Apoe2/2 mice
showed significant loss of synaptophysin-positive presynaptic terminals and MAP-2-positive neuronal dendrites in the neocortex
(Figs. 10A–D, 11) and hippocampus (data not shown) as they aged.
Likewise, there was an age-related disruption of neurofilamentpositive axons in the hippocampus of these mice (data not shown).
These findings are consistent with the results of Masliah et al. (1995).
To test whether there is an apoE isoform-specific effect on the
age-dependent neurodegeneration in Apoe2/2 mice, we analyzed
neuronal integrity in NSE-apoE3 and NSE-apoE4 mice on the
Apoe2/2 background at 3–4 and 7–9 months of age. We found that
apoE3 prevented the age-dependent degeneration of synaptophysinpositive presynaptic terminals and MAP-2-positive neuronal dendrites found in Apoe2/2 mice, whereas apoE4 did not (illustrated
in Fig. 10E–H; semiquantitative evaluation in Fig. 11). Likewise,
apoE3 prevented the age-dependent loss of neurofilamentpositive axons in the hippocampus, whereas apoE4 did not (data
not shown). By all measures of neuronal integrity examined,
NSE-apoE3 mice closely resembled wild-type mice. In contrast,
NSE-apoE4 mice, like Apoe2/2 mice, showed a significant loss of
synaptophysin-positive presynaptic terminals and MAP-2-positive
neuronal dendrites (Figs. 10G,H, 11) and a severe disruption of
hippocampal axons (data not shown) at 7–9 months of age.
The development of neurodegenerative changes in NSE-apoE4
mice was also clearly age-dependent, because significant deficits
were seen at 7–9 months but not at 3–4 months of age (Fig. 11).
Measurement of neocortical synaptophysin levels
The histopathological analysis was complemented by ELISA
measurements of synaptophysin in particulate fractions from
brain homogenates of NSE-apoE3 and NSE-apoE4 mice. After
injection of 18 mg/kg kainic acid, neocortical synaptophysin content was significantly lower in NSE-apoE4 mice than in NSEFigure 5. Neuronal labeling for apoE in NSE-apoE mice and a human AD case. Immunostaining with antibodies against human apoE (A–F, H, I ) or
mouse apoE (G) in NSE-apoE3 mice (A, D), NSE-apoE4 mice (B, E), and a human AD case (H, I ) showed prominent neuronal labeling for human apoE,
whereas mouse apoE in wild-type mice was detected primarily in astrocytes (G), which were identified by colabeling with anti-GFAP antibody (data not
shown). No apoE expression was found in Apoe 2/2 controls (C, F ). Scale bars: A–C, 25 mm; D–I, 15 mm.
Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology J. Neurosci., June 15, 1999, 19(12):4867–4880 4873
apoE3 mice (absorbance values at 492 nm: 125.3 6 3.4 for
NSE-apoE4 mice, 159 6 1.5 for NSE-apoE3 mice, p , 0.05, n 5
4/group, 5–6 months of age). Similarly, neocortical synaptophysin
content was significantly lower in untreated 9-month-old NSEapoE4 mice than in age-matched NSE-apoE3 mice (absorbance
values at 492 nm: 129.5 6 5.3 for NSE-apoE4 mice, 160.0 6 2.7
for NSE-apoE3 mice, p , 0.05, n 5 4/group).
Our data reveal that human apoE3 and apoE4 expressed at
similar levels in the brains of Apoe2/2 mice differ significantly in
their capacity to protect against excitotoxin-induced neurodegeneration and in their long-term effects on neuronal integrity.
Age-dependent neurodegeneration seen in Apoe2/2 mice was prevented by apoE3, but not apoE4. Excitotoxin-induced neurodegeneration, a key mechanism of neuronal injury in acute neurodegenerative processes, such as head trauma and stroke (Meldrum and
Garthwaite, 1990; Lipton and Rosenberg, 1994), was minimal in
the presence of apoE3 but severe in the presence of apoE4.
Consistent with this result, a recent study that examined transgenic
mice expressing apoE3 or apoE4 under the control of the human
APOE regulatory sequences (Sheng et al., 1998) showed that mice
expressing apoE3 had significantly smaller infarcts after cerebral
ischemia than mice expressing apoE4 at higher levels.
Figure 6. Colabeling of cells in NSE-apoE
mouse brains for human apoE and for the neuronal marker MAP-2. Brain sections were
double-immunolabeled with antibodies against
human apoE (green; A2, B2, C2) and antibodies
against MAP-2 (red; A1, B1, C1) and imaged by
confocal microscopy. Pseudocolored images depict colabeled neurons in neocortex of an NSEapoE3 (A, B) and of an NSE-apoE4 ( C) mouse.
Scale bars: A1, A2, 15 mm; B1, B2, C1, C2, 7 mm.
4874 J. Neurosci., June 15, 1999, 19(12):4867–4880 Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology
In the present study, we chose to assess neurodegeneration in
the brains of NSE-apoE mice by quantifying immunoreactivity
for the neuronal markers synaptophysin and MAP-2. There is
ample evidence that the loss of synaptophysin-positive presynaptic terminals, MAP-2-positive neuronal dendrites, and
neurofilament-positive axons are relevant indicators of neurodegenerative disease processes. For example, a number of studies
have reported a loss of synaptophysin immunoreactivity in AD
brains (Terry et al., 1991; Zhan et al., 1993; Dickson et al., 1995;
Sze et al., 1997), and this loss correlated well with the extent of
cognitive impairments (Terry et al., 1991; Sze et al., 1997). Other
studies have reported severely disrupted neurofilament-, or tau-,
immunoreactive axons (Kowall and Kosik, 1987; Masliah et al.,
1993) in AD brains, and a significant decrease of MAP-2 immunoreactive dendrites in the brains of patients with HIV-1 encephalitis (Masliah et al., 1992). Loss of MAP-2- and synaptophysinimmunoreactive neuronal structures has also been a sensitive and
reliable indicator of neuropathological changes in the brains of
diverse transgenic animal models (Toggas et al., 1994; Masliah et
al., 1997; Buttini et al., 1998).
Excitotoxic neuronal injury is mediated, at least in part, by the
release of reactive oxygen species (Michaelis, 1998). In cell cultures, apoE can protect neurons against oxidative insults, and
apoE3 has much stronger antioxidative properties than apoE4
(Miyata and Smith, 1996). Our study indicates that, in vivo, apoE3
also protects neurons more effectively than apoE4 against insults
presumed to involve oxidative stress. The neurodegeneration
seen in NSE-apoE4 mice after an excitotoxic challenge could
relate to the poor outcome of human APOE e4 carriers after head
trauma or stroke (Nicoll et al., 1996; Slooter et al., 1997), because
these CNS injuries are mediated, at least in part, by excitotoxic
mechanisms (Meldrum and Garthwaite, 1990).
The age dependence of the differential CNS effects of apoE3
and apoE4 identified in the current study is intriguing. The
Figure 7. Differential protective effects of
apoE3 and apoE4 after kainic acid challenge. Neocortical sections of wild-type (A,
B), Apoe 2/2 (C, D), NSE-apoE3 (E, F ),
and NSE-apoE4 (G, H ) mice injected with
18 mg/kg kainic acid were immunostained
for synaptophysin (A, C, E, G) or MAP-2
(B, D, F, H ) and imaged by confocal microscopy. Cases with severe damage were selected for illustration. Note the prominent
loss of neuronal structures in the neocortex
of Apoe 2/2 and NSE-apoE4 mice. In contrast, only minimal neurodegenerative
changes were seen in the neocortex of NSEapoE3 mice. Scale bar, 55 mm.
Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology J. Neurosci., June 15, 1999, 19(12):4867–4880 4875
preservation of neuronal structures in young NSE-apoE4 mice
indicates that apoE is not essential for normal neuronal development and that deficits related to apoE4 are strongly dependent on
age-related factors. Notably, a behavioral analysis of wild-type,
Apoe2/2, NSE-apoE3, and NSE-apoE4 mice revealed significant
impairments in learning, memory, and exploratory behavior in
female NSE-apoE4, but not NSE-apoE3, mice (Raber et al.,
1998), suggesting that the structural and molecular alterations
documented in the current study may have important functional
consequences. These results could relate to the increased susceptibility to AD associated with the APOE e4 allele in humans,
which also appears to be stronger in females (Farrer et al., 1997).
The reason for this gender bias remains to be determined.
Age-dependent neurodegenerative changes in Apoe2/2 mice
that do not express human apoE are a matter of controversy,
because they have been observed by some (Masliah et al., 1995)
Figure 8. Semiquantitative comparison of the effects of apoE3 and
apoE4 on kainic acid-induced neurodegeneration. Neocortical sections of
mice injected with saline (black bars), 18 mg/kg kainic acid (hatched bars),
or 25 mg/kg kainic acid (white bars) were immunolabeled for synaptophysin ( A) or MAP-2 ( B). Three groups of mice with each genotype were
treated with saline, 18 mg/kg kainic acid, or 25 mg/kg kainic acid,
respectively (number of mice in each group indicated in parentheses): 14
wild-type controls (5, 6, 3), 28 Apoe 2/2 (10, 12, 6), 14 NSE-apoE3 (4, 6,
4), and 16 NSE-apoE4 (4, 5, 7). The percentage area of neuropil occupied
by immunoreactive dendrites or presynaptic terminals was determined by
confocal microscopy and computer-aided image analysis. Significant
excitotoxin-induced neurodegeneration was detected in NSE-apoE4,
Apoe 2/2, and wild-type mice. NSE-apoE3 mice showed no significant
excitotoxin-induced loss of synaptophysin-positive presynaptic terminals
and showed significant loss of MAP-2-positive neuronal dendrites only at
the higher dose of kainic acid. Values are means 6 SEM. *p , 0.05, **p ,
0.01 versus saline-injected mice of the same genotype (Dunnett’s post hoc
test). In the hippocampus, presynaptic terminals were significantly decreased in kainic acid-injected (25 mg/kg) Apoe 2/2 and NSE-apoE4 mice,
and neuronal dendrites were significantly decreased in kainic acidinjected (18 mg/kg) wild-type, Apoe 2/2, and NSE-apoE4 mice when
compared with saline-injected controls of the same genotype (data not
shown). No significant decreases in presynaptic terminals or neuronal
dendrites were found in the hippocampus of kainic acid-injected (18 or 25
mg/kg) NSE-apoE3 mice (data not shown).
Figure 9. Differential effects of apoE3 and apoE4 on axonal structures
after kainic acid challenge. Hippocampal sections of 6-month-old wildtype (A), Apoe 2/2 (B), NSE-apoE3 ( C), and NSE-apoE4 ( D) mice
injected with 18 mg/kg kainic acid were immunostained for axonal neurofilaments and imaged by confocal microscopy. Note the prominent
disruption of neurofilament-positive axons in the hippocampus (CA1–
CA2 subfields) of Apoe 2/2 and NSE-apoE4 mice. Scale bar, 120 mm.
4876 J. Neurosci., June 15, 1999, 19(12):4867–4880 Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology
but not others (Anderson et al., 1998). The authors of the latter
study proposed that differences in mouse strains and/or origin of
the Apoe2/2 mice could account for this discrepancy. However,
this is unlikely, because we and others (E. Masliah, personal
communication) have observed age-dependent neurodegenerative changes in the brains of Apoe2/2 mice originating from the
same source and bred onto the same strain (C57BL/6J) as the
mice used by Anderson et al. (1998). It is conceivable that specific
dietary or other housing-related factors could increase agerelated stresses on neurons and thereby help reveal the lack of
neuroprotective apoE effects in some cohorts of aging Apoe2/2
mice. Potential differences in such environmental variables and in
methodological approaches will need to be scrutinized in the
future to resolve the divergent findings obtained in distinct
groups of Apoe2/2 mice.
There are differences in the brain cell-specific distribution of
endogenous mouse apoE in wild-type mice, of endogenous apoE
in humans, and of transgene-derived human apoE in NSE-apoE
mice. In wild-type mice, apoE mRNA and immunoreactivity have
been detected primarily in astrocytes, whereas neuronal apoE
labeling is more widespread and intense in human brains (Boyles
et al., 1985; Diedrich et al., 1991; Han et al., 1994; Bao et al., 1996;
Metzger et al., 1996). Neuronal expression of apoE mRNA has
recently been detected by in situ hybridization in the frontal
cortex and hippocampus of human brains, providing evidence
that human neurons are indeed capable of producing apoE (Xu et
al., 1999). Furthermore, striking increases in neuronal immunostaining for apoE have been documented after CNS injuries in
humans and rodents (Kida et al., 1995; Horsburgh and Nicoll,
1996). The detection of human apoE in the CSF (Fig. 3) and of
human apoE immunoreactivity in the neuropil (Fig. 4) of NSEapoE mice indicates that transgenic neurons secrete the human
apoE they produce, allowing for the interaction of transgenederived apoEs with all CNS cell types.
Figure 10. Differential effects of apoE3
and apoE4 on neuronal integrity in untreated Apoe 2/2 mice. Sections of neocortex from 7- to 9-month-old wild-type (A, B),
Apoe 2/2 (C, D), NSE-apoE3 (E, F ), and
NSE-apoE4 (G, H ) mice were immunostained for synaptophysin (A, C, E, G) or
for MAP-2 (B, D, F, H ) and imaged by
confocal microscopy. Cases with severe
damage were selected for illustration. Note
the prominent loss of immunolabeled neuronal structures in the neocortex of
Apoe 2/2 mice and NSE-apoE4 mice and
the normal appearance of corresponding
sections from wild-type and NSE-apoE3
mice. Qualitatively similar results were obtained for synaptophysin-positive presynaptic terminals in the hippocampus (data not
shown). Scale bar, 55 mm.
Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology J. Neurosci., June 15, 1999, 19(12):4867–4880 4877
Recently, we (T. Wyss-Coray, M. Buttini, R. E. Pitas, R. W.
Mahley, and L. Mucke, unpublished results) and others (Sun et
al., 1998) generated transgenic mice in which human apoE isoforms are expressed in astrocytes directed by the glial fibrillary
acidic protein promoter. Comparison of these models with the
NSE-apoE mice should allow us to assess the importance of the
cell type in which human apoE isoforms are produced.
No matter what cell type is used to express human apoE
isoforms in the brain, it is critical that the isoforms to be compared are expressed at similar levels and in a similar distribution
across different brain regions. As documented in Figures 2, 3, 5,
and 6, these requirements were clearly met in the current study.
Furthermore, in all the age groups tested, the extent of neuronal
damage did not differ significantly between Apoe2/2 mice from
the NSE-apoE3 line and age-matched Apoe2/2 mice from the
NSE-apoE4 line (data not shown), indicating that the lines were
well matched with respect to background genes.
We found no evidence that apoE expression had peripheral
effects in NSE-apoE3 or NSE-apoE4 mice. This is not surprising
because NSE-driven constructs are expressed primarily in the
CNS (Fig. 2A) (Forss-Petter et al., 1990; Mucke et al., 1994).
Therefore, the human apoE isoform-specific effects revealed by
the current study pertain primarily to CNS disorders. Our models
cannot determine whether human apoE3 and apoE4 have similar
differential effects in peripheral organs or whether such effects
might have indirect consequences for the nervous system. Answers to these questions can only be provided by related models
in which different human apoE isoforms are expressed in multiple
organs (Xu et al., 1996; Sullivan et al., 1997).
Although the findings we obtained in our NSE-apoE models
will need to be confirmed in other lines of apoE transgenic mice,
it is tempting to speculate that they may relate closely to the
effects of apoE isoforms in humans with AD. It is interesting in
this context that brains of AD patients carrying one or two
APOE e4 alleles show more severe neurodegeneration and less
dendritic arborization than brains of AD patients with two
APOE e3 alleles (Arendt et al., 1997). The potential relevance of
the NSE-apoE models to AD has also been highlighted by a
recent behavioral analysis that revealed age-dependent cognitive
deficits in female NSE-apoE4, but not NSE-apoE3, mice (Raber
et al., 1998).
In conclusion, we have demonstrated that distinct human apoE
isoforms differ significantly in their long-term effects on neuronal
integrity as well as in their ability to protect against excitotoxicity.
These differences in the neuroprotective capacities of apoE3 and
apoE4 could contribute to the increased susceptibility of human
APOE e4 carriers to AD and other types of CNS impairment.
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Figure 11. Semiquantitative comparison of apoE3 and apoE4 effects at
different ages. Neocortical sections of 3- to 4-month-old (A, C) and 7- to
9-month-old (B, D) mice were immunolabeled for synaptophysin (A, B) or
MAP-2 (C, D). Studies were performed on two groups of mice with each
genotype, 3–4 months or 7–9 months of age, respectively (number of mice
in each group indicated in parentheses): 7 wild-type controls (4, 3), 22
Apoe 2/2 (10, 12), 9 NSE-apoE3 mice (5, 4), and 14 NSE-apoE4 mice (5,
9). The percentage area of neuropil occupied by immunolabeled dendrites
or presynaptic terminals was determined by confocal microscopy and
computer-aided image analysis. In younger mice, the only significant
alteration detected was a rarefaction of dendrites in Apoe 2/2 mice ( B). By
7–9 months of age, both Apoe 2/2 and NSE-apoE4 mice had developed a
significant loss of immunopositive neuronal dendrites and presynaptic
terminals (B, D). In contrast, neuronal integrity in NSE-apoE3 mice was
similar to that of wild-type mice and significantly better than that of
NSE-apoE4 mice. Values are means 6 SEM. *p , 0.05, **p , 0.01 versus
wild-type (Dunnett’s post hoc test), 0p , 0.05 (Tukey-Kramer post hoc
test). Presynaptic terminals were also significantly decreased in the hippocampus of 7- to 9-month-old Apoe 2/2 and NSE-apoE4 mice ( p , 0.01
vs wild-type controls by Dunnett’s post hoc test) but not in age-matched
NSE-apoE3 mice (data not shown).
4878 J. Neurosci., June 15, 1999, 19(12):4867–4880 Buttini et al. • Isoform-Specific Effects of Human ApoE on Brain Pathology
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