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The Journal of Neuroscience, June 15, 1999, 19(12):4867-4880
Expression of Human Apolipoprotein E3 or E4 in the Brains of
Apoe
/ Mice: Isoform-Specific Effects on
Neurodegeneration
Manuel
Buttini1, 4,
Matthias
Orth1, 2,
Stefano
Bellosta1, 2,
Hassibullah
Akeefe1,
Robert E.
Pitas1, 2, 5,
Tony
Wyss-Coray1, 4,
Lennart
Mucke1, 4, and
Robert W.
Mahley1, 2, 3, 5
1 Gladstone Institute of Neurological Disease,
2 Cardiovascular Research Institute, and Departments of
3 Medicine, 4 Neurology, and
5 Pathology, University of California, San Francisco,
California 94141-9100
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ABSTRACT |
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 (Apoe
/) 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 Apoe/ 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 excitotoxin-induced neuronal damage. ApoE3, but
not apoE4, also protected against the age-dependent neurodegeneration
seen in Apoe/ 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
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INTRODUCTION |
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 Apoe/ 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 Apoe/ 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 A
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 Apoe/ mice using the
neuron-specific 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 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.
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MATERIALS AND METHODS |
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/ mice, NSE-apoE3 mice, and
NSE-apoE4 mice. NSE-apoE transgenic mice on the
Apoe/ 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/ 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/ mice
(C57BL/6J-Apoetm1Unc) from Jackson Laboratories (Bar
Harbor, ME) to generate NSE-apoE3 and NSE-apoE4 mice that were at least
75% C57BL/6J. Crosses of NSE-apoE3 or NSE-apoE4 with
C57BL/6J-Apoetm1Unc mice from Jackson Laboratories
also yielded nontransgenic Apoe/
littermates (n = 27). Comparison of the latter mice
with age-matched C57BL/6J-Apoetm1Unc mice from
Jackson Laboratories (n = 23) revealed no significant differences in any of the variables examined (data not shown). Therefore, these two cohorts of mice were combined
(Apoe/ 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 µl) was subjected to touchdown PCR (Hecker and Roux, 1996) in a total reaction volume of 25 µl with each primer (0.2 µM),
dNTPs (dATP, dCTP, dGTP, dTTP, 200 µM each), and 0.15 µl 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 acid-induced 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 
70°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 -actin mRNA [nucleotides 480-559 of
mouse -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 µg) hybridized to
32P-labeled antisense riboprobes was digested with 300 U/ml
RNase T1 (Life Technologies, Gaithersburg, MD) and 0.5 µg/ml RNase A (Sigma) in 100 µl 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 probe-specific signals with a phosphorimager
(FUJI-BasIII, Fuji, Tokyo, Japan); -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 NSE-apoE4, and eight
Apoe/ 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 µl
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 µg/ml), aprotinin (1 µg/ml), and complete inhibitor (2×, 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-µm-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 µg/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 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 anti-rat 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) anti-goat to detect antigen-bound anti-human
apoE or FITC-coupled anti-rabbit (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 µm
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 anti-GFAP).
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 × 140 µm. 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 edge-enhancement 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 × g
for 10 min at 4°C. The supernatant was then ultracentrifuged (100,000 × g for 1 hr at 4°C). The resulting pellet
(particulate fraction) was resuspended in 300-400 µl 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 µg 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 µg 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 ± 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.
| |
RESULTS |
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 (Apoe/)
background as described in Materials and Methods.
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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
5' to 3', 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 3'
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.
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Four groups of mice were analyzed in detail: wild-type mice,
Apoe/ mice, and
Apoe/ mice heterozygous for the
NSE-apoE3 transgene (NSE-apoE3 mice) or the NSE-apoE4 transgene
(NSE-apoE4 mice).
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
Apoe/ 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).
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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 = 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/
(lanes 7, 8) mice and analyzed by RPA. The
leftmost 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 = 9) and in corresponding groups of 3- to
4-month-old mice (n = 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 = 4) or NSE-apoE4 mice (n = 6) or from the midfrontal gyrus of humans without dementia
(n = 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.
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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
Apoe/ 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.
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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 = 2 mice/genotype, 50 µg protein/lane) and CSF (14 µl/lane) from
NSE-apoE3 and NSE-apoE4 mice. Human brain (occipital lobe) homogenate
(50 µg 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 ± 0.4 µg apoE/gm protein for NSE-apoE3 mice (n = 9), and 4.6 ± 0.5 µg apoE/gm protein for NSE-apoE4 mice
(n = 13). The apoE content in the CSF of mice from
both genotypes was 2.8 ng/µl (pooled from 6 mice/genotype).
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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 = 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 and NSE-apoE4 mice (Fig.
3A). No apoE was detected in the CSF of
Apoe/ 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).
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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/ 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 µm; all
other panels, 200 µm.
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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 
3/4), 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).
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Figure 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/ controls
(C, F). Scale bars: A-C, 25 µm;
D-I, 15 µm.
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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 NSE-apoE3 (A,
B) and of an NSE-apoE4 (C) mouse. Scale
bars: A1, A2, 15 µm; B1, B2, C1, C2, 7 µm.
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Comparison of the neuronal human apoE immunofluorescence signals in 3- to 4- and 7- to 9-month-old NSE-apoE3 and NSE-apoE4 mice
(n = 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 excitotoxin-induced
neurodegeneration in Apoe/ 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 Apoe/
background) with kainic acid. Control mice were injected with saline.
Kainic acid- and saline-injected
Apoe/ 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 excitotoxin-induced
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). Apoe/ 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-2-positive neuronal dendrites were observed in the hippocampus
(data not shown).
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Figure 7.
Differential protective effects of apoE3 and apoE4
after kainic acid challenge. Neocortical sections of wild-type
(A, B),
Apoe/ (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/ and NSE-apoE4 mice. In
contrast, only minimal neurodegenerative changes were seen in the
neocortex of NSE-apoE3 mice. Scale bar, 55 µm.
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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/ (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/, 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 ± 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/ and NSE-apoE4 mice, and
neuronal dendrites were significantly decreased in kainic acid-injected
(18 mg/kg) wild-type, Apoe/, 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).
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Figure 9.
Differential effects of apoE3 and apoE4 on axonal
structures after kainic acid challenge. Hippocampal sections of
6-month-old wild-type (A),
Apoe/ (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/ and NSE-apoE4 mice. Scale
bar, 120 µm.
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Differential effects of apoE3 and apoE4 on age-related
neurodegeneration in Apoe/ mice
To determine whether there is an age-related loss of neuronal
structures in Apoe/ mice, as has been
found by some (Masliah et al., 1995) but not by others (Anderson et
al., 1998), we analyzed neuronal integrity in
Apoe/ mice at 3-4 and 7-9 months of
age. Compared with age-matched wild-type controls,
Apoe/ 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
neurofilament-positive axons in the hippocampus of these mice (data not
shown). These findings are consistent with the results of Masliah et
al. (1995).
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Figure 10.
Differential effects of apoE3 and apoE4 on
neuronal integrity in untreated
Apoe/ mice. Sections of neocortex
from 7- to 9-month-old wild-type (A, B),
Apoe/ (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/ 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 µm.
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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/ (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/
mice (B). By 7-9 months of age, both
Apoe/ 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 ± 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/ 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).
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To test whether there is an apoE isoform-specific effect on the
age-dependent neurodegeneration in
Apoe/ mice, we analyzed
neuronal integrity in NSE-apoE3 and NSE-apoE4 mice on the
Apoe/ background at 3-4 and 7-9
months of age. We found that apoE3 prevented the age-dependent
degeneration of synaptophysin-positive presynaptic terminals and
MAP-2-positive neuronal dendrites found in
Apoe/ mice, whereas apoE4 did not
(illustrated in Fig. 10E-H; semiquantitative evaluation in Fig. 11). Likewise, apoE3 prevented the age-dependent loss of neurofilament-positive 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 Apoe/ 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 by ELISA
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 NSE-apoE3 mice (absorbance values at
492 nm: 125.3 ± 3.4 for NSE-apoE4 mice, 159 ± 1.5 for
NSE-apoE3 mice, p < 0.05, n = 4/group,
5-6 months of age). Similarly, neocortical synaptophysin content was
significantly lower in untreated 9-month-old NSE-apoE4 mice than in
age-matched NSE-apoE3 mice (absorbance values at 492 nm: 129.5 ± 5.3 for NSE-apoE4 mice, 160.0 ± 2.7 for NSE-apoE3 mice,
p < 0.05, n = 4/group).
| |
DISCUSSION |
Our data reveal that human apoE3 and apoE4 expressed at similar
levels in the brains of Apoe/ 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 Apoe/ 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.
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 synaptophysin-immunoreactive 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 4 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 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,
Apoe/, 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 4 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
Apoe/ mice that do not express human
apoE are a matter of controversy, because they have been observed by
some (Masliah et al., 1995) but not others (Anderson et al., 1998). The
authors of the latter study proposed that differences in mouse strains
and/or origin of the Apoe/ 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
Apoe/ 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 age-related stresses on
neurons and thereby help reveal the lack of neuroprotective apoE
effects in some cohorts of aging
Apoe/ 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 Apoe/ 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 NSE-apoE mice
indicates that transgenic neurons secrete the human apoE they produce,
allowing for the interaction of transgene-derived apoEs with all CNS
cell types.
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 Apoe/ mice from
the NSE-apoE3 line and age-matched
Apoe/ 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 4 alleles show more severe
neurodegeneration and less dendritic arborization than brains of AD
patients with two APOE 3 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
4 carriers to AD and other types of CNS impairment.
| |
FOOTNOTES |
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.
| |
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TOP
ABSTRACT
INTRODUCTION
