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From the Experimental Genetics Group, Center for Human Genetics, Flemish Institute for Biotechnology, Leuven, Belgium
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 allele of
apolipoprotein E has been associated with increased plaque
load3-5
and with early onset of AD-related
neurofibrillary changes in young individuals.6
Recently,
genetic studies reported polymorphisms in the promoter region of the
human ApoE gene associated with AD.7-10
These
might pertain to the observation that, in the brains of AD patients,
ApoE messenger RNA (mRNA) was increased.11
In
addition to its well-documented role in AD, the ApoE4 allele
has been implicated in poorer neurological recovery to head injury,
cerebral hemorrhage, and cognitive status after cardiac bypass
surgery.12-17
These experiments provide epidemiological
evidence for the close relationship between ApoE and AD or between ApoE
and the outcome in nervous system injury, but do not provide an
explanation for its mechanism of action within the nervous
system. The demonstration that the mainly astrocytic protein ApoE can also be found in human neurons might be important in assessing its role within the nervous system. Immunocytochemically, ApoE was demonstrated in human hippocampal neurons of AD patients and aged individuals18 and in cortical neurons of elderly.19 Recently, human brain neurons have been shown to synthesize ApoE. In situ hybridization revealed ApoE mRNA in the neurons of the CA1 to CA4 region of the hippocampus, the granule cell layer of the dentate gyrus, and many neurons in the frontal cortex.20 In addition, transgenic mice that overexpress large fragments of the human ApoE locus, including the human ApoE promoter, also showed neuronal expression of human ApoE.21-23 It is interesting that the regions where neuronal ApoE was present are most vulnerable in developing neurofibrillary tangles.20,24 Also, human neuroblastoma cells were shown to synthesize ApoE mRNA.25,26 In rodent brain, ApoE has been demonstrated in central neurons only after experimental brain injury.27-30 Numerous cell culture experiments have demonstrated receptor-mediated uptake of ApoE in neurons, and large effects on neuritic outgrowth and morphology were found.31-37 In vitro, protein-protein interactions of ApoE with intracellular microtubule-associated proteins (MAP2c, tau) were observed.2,38 These results support an intracellular role for ApoE in neuronal pathology in AD and suggest that ApoE might be more directly involved in disruption of the neuronal cytoskeleton.
To study the repercussions of ApoE expression in different cell types in the brain, we have generated 25 independent founder transgenic mice that overexpress human ApoE4 in neurons and/or astrocytes. We have used four different gene promoter constructs derived from either the mouse Thy1 gene,39 the human GFAP gene,40 the human PDGF-ß gene,41 or the mouse PGK gene.42 The initial phenotypic characterization demonstrated strong neuronal expression in the hippocampus and cortex of Thy1-ApoE4 and PDGF-ApoE4 transgenic mice, whereas the PGK-ApoE4 transgenic mice showed neuronal expression over the entire brain. As expected, in the GFAP-ApoE4 transgenic mice, expression was restricted to astroglia. Surprisingly, with a panel of protein tau-specific antibodies, hyperphosphorylation of the microtubule-associated protein tau became evident in the transgenic mice that overexpressed human ApoE4 in neurons. This might provide a clue to how ApoE could influence or disturb neuronal cytoskeletal stability.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All constructs were based on a 5.5-kb
BamHI-HindIII fragment of the human
ApoE4 gene (Figure 1)
. The
mouse thy1 gene, cloned as an 8.1-kb EcoRI
genomic fragment,39
was adapted by replacing a 1.5-kb
BanHI-XhoI fragment with a synthetic
XhoI oligonucleotide adapter, in which the human ApoE4
BamHI-HindIII fragment was ligated. The
PDGF gene promoter41,43
and the GFAP
gene promoter40,44
were combined with the 1.5-kb
EcoRI-XhoI fragment containing the mouse
thy1 gene 3'UTR with polyadenylation signal. For the
PGK-ApoE4 construct, the 5.5-kb human ApoE4 fragment was
ligated in the PstI site of a PGK expression
cassette.42,45
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Routinely, transgenic offspring was identified by polymerase chain reaction, using primers in exon 4 of the human ApoE4 gene as follows: forward primer, 5'-GCGGGCACGGCTGTCCAAG (P78); reverse primer, 5'-GGGGTGGCGTGGGGTCGCAT (P79). Mouse tail biopsies were digested overnight at 55°C with 1 mg/µl proteinase K in 50 mmol/L Tris-HCI (pH 8.5), 1 mmol/L EDTA, 0.5% sodium dodecyl sulfate, 0.1 mol/L NaCl. The enzyme was inactivated by boiling for 10 minutes, and diluted samples were analyzed by polymerase chain reaction with the following program: 2 minutes at 95°C, followed by 30 cycles of 1 minute at 95°C, 1 minute at 60°C, 1 minute at 72°C, and a final extension of 15 minutes at 72°C.
Histology and Immunohistochemistry
Anesthetized mice were perfused with phosphate-buffered saline followed by 4% paraformaldehyde. Whole brain was fixed for 16 hours in 4% paraformaldehyde at 4°C and washed in phosphate-buffered saline, dehydrated, and embedded in paraffin for sectioning (5–7 µm). Alternatively, vibratome sections of 40 µm were cut and transferred to microtiter wells in phosphate-buffered saline containing 0.5% sodium azide at 4°C.
Paraffin sections were routinely stained with hematoxylin-eosin (H&E), cresylviolet (Nissl stain), and Garvey silver stain by standard procedures. Immunohistochemical staining of sections was performed by standard procedures with commercially available antibodies: ApoE (Dako, Glostrup, Denmark), GFAP (Dako), ubiquitin (Dako), AT8 (Innogenetics, Ghent, Belgium), PHF1 and Alz50 (P. Davies), and B19 (J. P. Brion). Vibratome sections were treated with 0.6% (w/v) hydrogen peroxide to quench the endogenous peroxidase activity. After rinsing, sections were incubated in Tris-buffered saline, containing 10% goat serum and 0.2% Triton-X-100 for 2 hours, before incubation with antibodies against ApoE (1:10,000, overnight at room temperature). For immunohistochemical staining with B19 (1:500), the detergent was omitted. Secondary antibody was biotinylated goat-anti-rabbit immunoglobulin G (IgG) followed by StreptABComplex/HRP (Dako). Final staining was developed with 0.075% 3,3-diaminobenzidine and 0.01% (w/v) hydrogen peroxide. Staining with the monoclonal antibodies AT8 (1:80), PHF1 (1:500), and Alz50 (1:50) was performed with the Dako ARK staining method, by the instructions of the manufacturer. Sections were mounted on gelatin-coated glass slides, counterstained with hematoxylin, dehydrated, and mounted.
In Situ Hybridization
The sense and antisense human ApoE4 probes were synthesized from a pBluescript (SK-) vector (Stratagene, La Jolla, CA) in which we cloned a 161-bp EcoNI/Eco47III restriction fragment from the human ApoE4 gene. The plasmid was linearized with either EcoRI or ClaI and transcribed with T7 and T3 RNA polymerase, respectively, in the presence of [35S]UTP. Paraffin sections (6 µm) were transferred on silanylated glass slides, dewaxed, and rehydrated through an ethanol series. Sections were digested with proteinase K (20 µg/ml), postfixed in 4% paraformaldehyde, and treated with 0.25% acetic anhydride in 0.1 mol/L triethanolamine-HCl. Sections were hybridized overnight in 50% deionized formamide, 0.3 mol/L NaCl, 20 mmol/L Tris-HCl, 5 mmol/L EDTA (pH 8.0) with 10% dextran sulfate, 1x Denhardt’s solution, 0.5 mg/ml yeast RNA, and 10 mmol/L dithiothreitol and supplemented with the appropriate radiolabeled riboprobe. After stringency washes and ribonucleaseA treatment, sections were dehydrated, and dipped in photographic emulsion (LM-1, Amersham) and exposed for 1 week.
Analysis of RNA and Protein
Brains of 6-week-old transgenic offspring were removed from the skulls as quickly as possible; for each, one hemisphere was used for analysis of ApoE mRNA, and the other was used for analysis of ApoE protein expression. Analysis of RNA and protein was done as previously described.39 ApoE protein was detected with a polyclonal antibody to human ApoE4 (Dako).
In brains from 3- to 24-month-old transgenic offspring, one hemisphere was fixed in 4% paraformaldehyde and processed for immunohistochemistry, and the other was used for Western blot analysis of protein tau. Samples were homogenized in 10 vol of buffer consisting of 0.1 mol/L 2-(N-morpholino)ethanesulfonic acid (Mes), pH 6.4, 0.5 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L ethylene glycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Just before use, the following proteinase and phosphatase inhibitors were added: 1 mmol/L dithiothreitol, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 200 µmol/L phenylmethylsulfonyl fluoride, 20 mmol/L NaF, 100 mmol/L Na3VO4, 1 µmol/L okadaic acid, 1 mmol/L EDTA, 5 µg/ml soybean trypsin inhibitor, 1% sodium deoxycholate, 1% Trition-X-100, and 0.1% sodium dodecyl sulfate (final concentrations). The homogenized suspension was centrifuged for 30 minutes at 100,000 x g at 4°C. Supernatants were stored at -70°C until use. Protein concentration was measured with the BioRad Dc protein Assay (BioRad, Hercules, CA). Before loading on the gel, 2% sodium chloride and 5% ß-mercaptoethanol were added. Samples were heated at 100°C for 10 minutes in tightly capped tubes, chilled on ice for 30 minutes, and centrifuged for 15 minutes at 12,000 x g at 4°C. To the supernatants, 2% sodium dodecyl sulfate, 1% ß-mercaptoethanol, and 10% glycerol blue (final concentrations) were added. After heating at 95°C for 10 minutes, proteins were separated by denaturing sodium dodecyl sulfate-polyacrylamide gels (NOVEX, Frankfurt-au-Main, Germany). Western blotting was performed with monoclonal antibodies PHF1, AT8, AT180, and TAU5. The monoclonal antibodies AT8 and AT180 (Innogenetics) recognize protein tau phosphorylated at serine-202 and threonine-205 (AT8), and threonine-231 (AT180). Monoclonal antibody PHF-1 (kindly provided by P. Davies) is specific for protein tau phosphorylated at serine-396 and serine-404. Total tau protein was detected with monoclonal antibody TAU5 (Pharmingen, San Diego, CA), which recognizes a phosphorylation-independent epitope of tau protein. Dephosphorylation of protein tau was performed with 300 U/ml alkaline phosphatase (Boehringer Mannheim, Mannheim, Germany), incubated at 37°C for 1 h.
Quantitative analysis of Western blots, assessed by densitometric scanning, was normalized, and 3 to 4 mice per transgenic strain and per age group were analyzed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice were generated that overexpress human ApoE4 under
the control of well-characterized promoters, derived from the following
four genes: the mouse Thy1 gene, the human PDGF
gene, the human GFAP gene, and the mouse PGK
gene. The constructs are schematically represented in Figure 1A
, and
the known expression patterns of the gene promoters are summarized in
Table 1
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Analysis of Expression of Human ApoE4
Expression of human ApoE4 in brain was demonstrated and measured
by Northern and Western blotting in F1 offspring (2 months old), from
all founders. Comparative analysis of expression of human ApoE4 mRNA
and protein in brain from the selected transgenic mouse strains is
shown in Figure 1, B and C
, respectively. Northern blotting
demonstrated expression in all tissues examined in the PGK-ApoE4
transgenic mice, whereas in situ hybridization showed
expression already in postimplantation embryos at E5.5.
Quantitation of relative levels of ApoE4 mRNA by densitometric scanning, showed that expression levels in the tae-II and tae-XIII transgenic mice were, respectively, 3- and 4.4-fold higher than in pae-II transgenic mice. In the gae-I transgenic mice, ApoE4 expression levels were 4.5-fold higher than in pae-II transgenic mice.
The spatial distribution of human ApoE4 mRNA was visualized by in
situ hybridization in brain of adult transgenic mice. Expression
patterns resulting from the different gene promoters were as
expected.40,41,43,44,46-52
Strong hybridization signals
in pyramidal cells of the hippocampus, in the granular cells of the
dentate gyrus, and in the deeper layers of the cerebellar cortex were
detected in the brains of Thy1-ApoE4 mice (Figure 2, A and C)
. Expression was further
evident in neurons of the amygdala and striatum. Signals were most
restricted to neuronal cell bodies, with minor hybridization signals in
the proximal ends of some neurites. PDGF-ApoE4 transgenic
mice strongly expressed in the neuronal cell bodies of the cortex, in
pyramidal cells of the hippocampus, and in granular cells of the
dentate gyrus, but only weakly in amygdala (Figure 2, F and H)
. The
PGK-ApoE4 mice showed diffuse expression in cells over the entire
brain, with pyramidal and granular cells of the hippocampus and cells
in the choroid plexus displaying stronger signals (Figure 2, G and I)
.
In contrast, but as expected, GFAP-ApoE4 mice expressed the
transgene strongly in astrocytes (Figure 2, B and D)
. The presence of
weak signals in layer II and V neurons in the cortex could not be
excluded.
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was similar to the distribution of its
mRNA in all mice analyzed from the seven transgenic strains. In the
Thy1-ApoE4, PDGF-ApoE4, and PGK-ApoE4 transgenic mice,
we observed somatodendritic staining of neurons (Figure 3, A, C, D, F, G, and J)
, whereas in the GFAP-ApoE4 transgenic mice, ApoE
immunoreaction was restricted to astrocytes (Figure 3, H and I)
. In the
Thy1-ApoE4 transgenic mice, a granular staining pattern, probably
corresponding to vesicular structures, was present in some dendrites
(Figure 3D)
.
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All transgenic mice from all constructs appeared normal during the first 2 to 3 months of life. Thereafter, many mice progressively manifested motor problems accompanied by muscle wasting, loss of total body weight, and premature death. These signs were evident in the five transgenic strains with Thy1-ApoE4 (3 to 12 months old), PDGF-ApoE4, and PGK-ApoE4 constructs (12 months or older).
Thy1-ApoE4 transgenic mice with highest expression levels
progressively developed the most severe phenotype. At around 6 months
of age, about 60% of the mice in strain tae-II displayed bleeding
excoriations on the eyelids, which appeared swollen. Close observation
revealed these to result from scratching with the hind legs. Homozygous
animals developed the phenotype at an earlier age than heterozygous
animals of the same strain, housed in the same conditions. Muscle
wasting and loss of bodyweight (30%) accompanied these symptoms, most
likely due to the progressive inability to climb to the roof of the
cage for feeding. This was further reflected in the increased mortality
of the Thy1-ApoE4 transgenic mice: at 6 months, already 66% of
transgenic mice in the highest expressing strain (tae-XIII) and 25% in
the lower expressing Thy1-ApoE4 strain (tae-II) succumbed (Figure 4)
.
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. Hyperphosphorylation of Protein tau in Thy1-ApoE4 Transgenic Mice
We investigated protein tau phosphorylation in mice transgenic for human apolipoprotein E4 by Western blotting of brain homogenates. tau proteins are separated as a complex set of protein species reflecting both the expression of several isoforms and the presence of differentially phosphorylated species.53
Protein tau phosphorylation was comparatively analyzed with four
specific monoclonal antibodies, ie, AT8, AT180, PHF1, and TAU5. Western
blot analysis of brain homogenates of transgenic mice showed that the
microtubule-associated protein tau became hyperphosphorylated in mice
expressing ApoE4 in neurons, which was demonstrated in two independent
Thy1-ApoE4 transgenic lines. An increase in protein tau
phosphorylation appeared in Thy1-ApoE4 line 13 (tae-XIII)
transgenic mice of 3 months and was very prominent in mice of 18 months
(Figure 5A)
. In line tae-II, with lower
neuronal expression levels, increased protein tau phosphorylation
appeared when mice were 7 months old, indicating that
hyperphosphorylation of protein tau correlated with neuronal ApoE4
expression levels. The marked decrease in electrophoretical mobility of
the immunoreactive protein tau isoforms, following immunoblotting with
TAU5 antibody, confirmed the increase in phosphorylation of protein tau
in the brain of Thy1-ApoE4 transgenic mice, demonstrated with
antibodies AT8, AT180, and PHF1 (Figure 5A)
. Dephosphorylation of
protein tau by pretreatment of brain extracts with alkaline phosphatase
reduced or abolished the immunoreaction of the slower-migrating
isoforms, detected with antibodies AT8 and TAU5 (Figure 7)
.
Phosphorylation of protein tau was variable at all age groups, but was
always higher in transgenic mice relative to age-matched Wt mice.
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. The 60-kd protein tau band, detected by the monoclonal
antibody TAU5, was threefold more intensive in tae-II transgenic mice
relative to age-matched Wt mice, when mice were 7 or 14 months old
(Figure 8)
, whereas total amounts of protein tau were not different
(Figure 8)
.
|
. Neither in GFAP-ApoE4
mice of two years old (Figure 6)
nor in PGK-ApoE4 mice of 12 to 17
months old was an increase in protein tau phosphorylation ever
observed.
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Histological and immunohistochemical analysis was performed on 28 transgenic and 11 Wt mice between 3 and 23 months old. The histological repercussion of ApoE4 overexpression was minimal as judged by H&E staining of brain sections of transgenic mice and age-matched Wt mice. The brain appeared microscopically normal and did not show signs of deviating architecture or neuronal loss.
Astrogliosis, assessed by immunostaining for GFAP, was evident in the
brain of older ApoE4-transgenic mice (Figure 9, A–F)
. Gliosis was most
prominent in the neocortex, hippocampus, and amygdala of transgenic
mice with highest neuronal expression levels, ie, in the Thy1-ApoE4 and
PDGF-ApoE4 mice (Figure 9, A, B, and F)
.
In the GFAP-ApoE4 mouse, only minimal gliosis was present (Figure 9D)
.
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. The inclusions were rounded or more angular in shape.
Immunohistochemical staining with B19, a polyclonal antiserum
recognizing protein tau independent of its phosphorylation state,
showed widespread axonal and somatodendritic staining in white matter
fiber tracts, neocortex, hippocampus, and thalamus, which was not
different in Thy1-ApoE4 transgenic mice relative to Wt mice
(Figure 10A)
. This staining pattern was also similar to the one
observed in rat brain.54
Staining with the
phosphate-dependent monoclonal antibody AT8 revealed reactivity mainly
in the neocortex, hippocampus, and amygdala of Thy1-ApoE4
transgenic mice. Staining was most intense in old Thy1-ApoE4
transgenic mice (12–18 months), compared with age-matched Wt mice
(Figure 10, B and C)
. The
phosphorylation-dependent monoclonal antibody PHF1 stained neurons in
the neocortex, thalamic nuclei, and the CA3 and CA4 regions of the
hippocampus. Staining was again most intense in old Thy1-ApoE4
transgenic mice compared with age-matched Wt mice. Staining with the
conformation-dependent monoclonal antibody Alz50 remained negative both
in Wt and Thy1-ApoE4 transgenic mice. Neither immunohistochemistry
nor silver staining revealed any neurofibrillary tangles or other types
of neurofibrillary inclusions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In three independent transgenic mouse strains, neuronal expression of human ApoE4 resulted in protein tau hyperphosphorylation. The level of neuronal expression, in combination with aging, appeared to be the most important determining factors. Offspring of the highest expressing Thy1-ApoE4 transgenic line (line tae-XIII) displayed protein tau hyperphosphorylation when 3 months old, whereas mild overexpression, ie, in line pae-II, resulted in hyperphosphorylation only when mice were 2 years old. Within a particular age group of Thy1-ApoE4 transgenic mice, protein tau hyperphosphorylation was somewhat variable, as reflected by the levels of the immunoreactive isoforms detected in Western blotting with specified monoclonal antibodies. Using the polyclonal phosphorylation-independent antibody B19, the somatodendritic localization of protein tau in cortical and hippocampal neurons was similar in old Thy1-ApoE4 transgenic and Wt mice. This result is in agreement with a previous study in rats, showing phosphorylated tau in the soma and dendrites of neurons in adult brain.54 In addition, the brain of Thy1-ApoE4 transgenic mice, shown to exhibit increased protein tau phosphorylation on Western blotting, also showed increased immunoreactivity for phosphorylated tau with antibodies AT8 and PHF1 on brain sections.
The remarkable phenotypic parameter of all transgenic mice that express ApoE4 in neurons, ie, protein tau hyperphosphorylation, relates to in vitro studies illustrating isoform-specific interactions between ApoE and protein tau.2 Our results demonstrate that, under specified conditions, ApoE4 can cause protein tau to become hyperphosphorylated directly, in vivo. Whether this is through a direct molecular interaction or indirectly, remains at present unclear.
Transgenic mice expressing ApoE4 in neurons under control of the neuron-specific enolase promoter were reported to have difficulties with learning in water maze tasks and with exploratory behavior at 6 months.55 Humanized transgenic mice carrying genomic sequences for ApoE have been generated, showing neuronal expression of ApoE in addition to glial expression.21-23 It would be interesting to know whether these mice would also show protein tau hyperphosphorylation at an older age. In the PDGF-ApoE4 transgenic mice, which have lower expression levels, tau hyperphosphorylation and motor impairments appeared only when mice were around 2 years old.
Other transgenic mice generated in our laboratory, expressing very high levels of unrelated proteins under control of the same Thy1 gene promoter, did not show protein tau hyperphosphorylation and remained completely normal throughout life. Therefore, we exclude the possibility of a general neurotoxic effect of overexpressing high levels of any protein in central neurons, under control of the Thy1 gene promoter.
Protein tau hyperphosphorylation has been demonstrated in mice lacking ApoE,56,57 although conflicting results have been reported.58 Because brain endogenous mouse ApoE levels remained unchanged in transgenic mice expressing human ApoE4 in neurons, the observed protein tau hyperphosphorylation cannot be attributed to a down-regulation or absence of endogenous mouse ApoE.
In apolipoprotein E transgenic mice generated with the same GFAP promoter as used here, ApoE proteins were secreted in high-density like lipoprotein particles.59 The present results suggest that expression of ApoE4 in glial cells, secretion, and eventual uptake by neurons were not a sufficient mechanism to cause protein tau hyperphosphorylation in neurons. Expression in neurons in combination with aging appeared to be the important factors.
Ubiquitin immunoreactivity has been associated with hyperphosphorylated protein tau in AD.60 In our experimental models, ubiquitin-positive inclusions were demonstrated in the brain of transgenic Thy1-ApoE4 and PDGF-ApoE4 mice, which were not found in Wt or GFAP-ApoE4 mice. However, silver staining or immunohistochemistry for protein tau did not reveal intraneuronal tangles or abnormal inclusions. In agreement with these results, neurofibrillary tangle formation could not be demonstrated in mouse models showing hyperphosphorylation of protein tau, not even when mice were 19 months old61-63 or in mice overexpressing and hyperphosphorylating human protein tau isoforms.64-66 In addition, it has been shown that AT8 immunoreactivity precedes tangle formation in AD and natural animal models such as aging sheep and goats.67,68
Reactive astrocytes reflect general central nervous system injury.69 Astrogliosis in the neocortex, hippocampus, and amygdala was strongest in Thy1-ApoE4, PDGF-ApoE4, and PGK-ApoE4 mice and absent in Wt mice. Only mild astrogliosis was present in GFAP-ApoE4 mice, and, despite higher expression levels than in the Thy1-ApoE4 mice, no signs of protein tau hyperphosphorylation were ever noted, at any age.
To explain the genetic association of ApoE4 to AD, two types of mechanisms have been proposed. First, ApoE could function as a "pathological chaperone," affecting clearance of ß-amyloid and causing amyloid deposition.70-72 We obtained no evidence for the presence of amyloid plaques in any of our ApoE4 transgenic mice, which of course might be because only endogenous mouse APP is present, which is less amyloidogenic.39 The second hypothesis states that ApoE interacts with the microtubule-associated protein tau, thereby altering its phosphorylation state, and hence is involved in stabilizing the neuronal cytoskeleton.2,73 The results presented here support this hypothesis. However, the route by which ApoE gains access to the neuronal cytoplasm has been the major criticism against this hypothesis and subject of much speculation. Most recently, it was shown that direct expression of ApoE in the cytosol of Neuro-2a cells is toxic.74 The ApoE4 transgenic mice are, however, not expected to express ApoE directly in the cytosol, and we also did not observe obvious signs of neurotoxicity. In addition, the hyperphosphorylation of protein tau was not restricted to brain regions expressing human ApoE as shown by immunohistochemistry. Therefore, we favor a mechanism involving an indirect interaction between ApoE and protein tau. In addition, this implies that hyperphosphorylation of protein tau is not simply a nonspecific downstream event marking degeneration.
Although great caution must be taken in extrapolating findings
from transgenic mice to complex human neurodegenerative diseases such
as AD, the results presented here might be relevant for the
pathological process in AD. An obvious question is whether expression
of the ApoE3 isoform in neurons, to the same levels as ApoE4, would
have the same or other effects. Reconciling two different hypotheses,
we propose that the phenotype we observed is not typical for ApoE4, but
will be observed with all ApoE isoforms. Recent in situ
hybridization experiments on human brain tissue and brain tissue of
humanized transgenic lines carrying genomic sequences for ApoE
demonstrated that low levels of human ApoE are expressed in neurons,
independently of the ApoE isotype,20-23
suggesting that
neuronal synthesis of ApoE is typical for and caused by regulatory
sequences in the human ApoE gene. In addition, polymorphisms
in the promoter region of the ApoE gene have been associated
with AD,7-10,75-77
and many of them were correlated with
increased ApoE expression.10,11,76,78
It is important that
tight linkage of ApoE gene promoter polymorphisms with the
4 allele was demonstrated.7,8,79
Although direct
evidence is lacking at this moment, combined with our observation that
neuronal expression of ApoE results in hyperphosphorylation of protein
tau, it is tempting to speculate that quantitative differences in ApoE
expression in neurons might be related to promoter polymorphisms and
that altered neuronal expression of ApoE would be genetically
associated with the 4 allele. This not only points to an important
function for neuronal ApoE but could also provide an alternative
explanation for the observed association of the 4 allele of
ApoE with AD. To prove this last hypothesis, a quantitative
analysis is needed of ApoE expression in neurons, related to the
different ApoE promoter polymorphisms and different ApoE
alleles in AD and controls. Obviously, this hypothesis does not exclude
additional effects, ie, a different extracellular role of the different
ApoE isoforms on neurons, as indicated by cell culture
experiments.31-37,80-82
In conclusion, we demonstrated that expression of human ApoE4 in neurons resulted in hyperphosphorylation of the microtubule-associated protein tau in vivo, as opposed to expression in non-neuronal cells. We also showed that the neuronal expression levels of human ApoE, as well as aging, are major factors for hyperphosphorylation of protein tau to become evident. Although many different aspects still need to be addressed, as discussed, the current transgenic mice offer the opportunity to investigate the interactions between ApoE and protein tau.
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Acknowledgements |
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Footnotes |
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Supported by the Fonds voor Wetenschappelijk Onderzoek (FWO), the Interuniversity-Network for Fundamental Research (IUAP), by the special Action Program for Biotechnology of the Flemish government (VLAB/IWT, COT-008), by the Rooms fund, and by KULeuven Research fund.
D. Moechars’ present adress: Janssens Research Foundation, B-2340 Beerse, Belgium.
Accepted for publication November 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice. EMBO J 1996, 15:1265-1274[Medline]
2-macroglobulin gene. J Biol Chem 1995, 1978, 270:19778-19785
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 F. M. Harris, W. J. Brecht, Q. Xu, R. W. Mahley, and Y. Huang Increased tau Phosphorylation in Apolipoprotein E4 Transgenic Mice Is Associated with Activation of Extracellular Signal-regulated Kinase: MODULATION BY ZINC J. Biol. Chem., October 22, 2004; 279(43): 44795 - 44801. [Abstract] [Full Text] [PDF]
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Q. Xu, W. J. Brecht, K. H. Weisgraber, R. W. Mahley, and Y. Huang Apolipoprotein E4 Domain Interaction Occurs in Living Neuronal Cells as Determined by Fluorescence Resonance Energy Transfer J. Biol. Chem., June 11, 2004; 279(24): 25511 - 25516. [Abstract] [Full Text] [PDF]
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 J. Raber Androgens, ApoE, and Alzheimer's Disease Sci. Aging Knowl. Environ., March 17, 2004; 2004(11): re2 - re2. [Abstract] [Full Text] [PDF]
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W. J. Brecht, F. M. Harris, S. Chang, I. Tesseur, G.-Q. Yu, Q. Xu, J. Dee Fish, T. Wyss-Coray, M. Buttini, L. Mucke, et al. Neuron-Specific Apolipoprotein E4 Proteolysis Is Associated with Increased Tau Phosphorylation in Brains of Transgenic Mice J. Neurosci., March 10, 2004; 24(10): 2527 - 2534. [Abstract] [Full Text] [PDF]
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F. M. Harris, I. Tesseur, W. J. Brecht, Q. Xu, K. Mullendorff, S. Chang, T. Wyss-Coray, R. W. Mahley, and Y. Huang Astroglial Regulation of Apolipoprotein E Expression in Neuronal Cells: IMPLICATIONS FOR ALZHEIMER'S DISEASE J. Biol. Chem., January 30, 2004; 279(5): 3862 - 3868. [Abstract] [Full Text] [PDF]
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F. M. Harris, W. J. Brecht, Q. Xu, I. Tesseur, L. Kekonius, T. Wyss-Coray, J. D. Fish, E. Masliah, P. C. Hopkins, K. Scearce-Levie, et al. Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice PNAS, September 16, 2003; 100(19): 10966 - 10971. [Abstract] [Full Text] [PDF]
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 K.R. Bales, J.C. Dodart, R.B. DeMattos, D.M. Holtzman, and S.M. Paul Apolipoprotein E, Amyloid, and Alzheimer Disease Mol. Interv., October 1, 2002; 2(6): 363 - 375. [Abstract] [Full Text] [PDF]
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I. Dewachter, D. Reverse, N. Caluwaerts, L. Ris, C. Kuiperi, C. Van den Haute, K. Spittaels, L. Umans, L. Serneels, E. Thiry, et al. Neuronal Deficiency of Presenilin 1 Inhibits Amyloid Plaque Formation and Corrects Hippocampal Long-Term Potentiation But Not a Cognitive Defect of Amyloid Precursor Protein [V717I] Transgenic Mice J. Neurosci., May 1, 2002; 22(9): 3445 - 3453. [Abstract] [Full Text] [PDF]
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 C. Lesuisse, G. Xu, J. Anderson, M. Wong, J. Jankowsky, G. Holtz, V. Gonzalez, P. C. Y. Wong, D. L. Price, F. Tang, et al. Hyper-expression of human apolipoprotein E4 in astroglia and neurons does not enhance amyloid deposition in transgenic mice Hum. Mol. Genet., October 1, 2001; 10(22): 2525 - 2537. [Abstract] [Full Text] [PDF]
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R. L. Raffai, L.-M. Dong, R. V. Farese Jr., and K. H. Weisgraber Introduction of human apolipoprotein E4 ""domain interaction"" into mouse apolipoprotein E PNAS, September 5, 2001; (2001) 201279298. [Abstract] [Full Text] [PDF]
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Y. Huang, X. Q. Liu, T. Wyss-Coray, W. J. Brecht, D. A. Sanan, and R. W. Mahley Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons PNAS, July 5, 2001; (2001) 151254698. [Abstract] [Full Text] [PDF]
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I. Tesseur, J. Van Dorpe, K. Bruynseels, F. Bronfman, R. Sciot, A. Van Lommel, and F. Van Leuven Prominent Axonopathy and Disruption of Axonal Transport in Transgenic Mice Expressing Human Apolipoprotein E4 in Neurons of Brain and Spinal Cord Am. J. Pathol., November 1, 2000; 157(5): 1495 - 1510. [Abstract] [Full Text] [PDF]
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J. Van Dorpe, L. Smeijers, I. Dewachter, D. Nuyens, K. Spittaels, C. Van den Haute, M. Mercken, D. Moechars, I. Laenen, C. Kuiperi, et al. Prominent Cerebral Amyloid Angiopathy in Transgenic Mice Overexpressing the London Mutant of Human APP in Neurons Am. J. Pathol., October 1, 2000; 157(4): 1283 - 1298. [Abstract] [Full Text] [PDF]
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K. Spittaels, C. Van den Haute, J. Van Dorpe, H. Geerts, M. Mercken, K. Bruynseels, R. Lasrado, K. Vandezande, I. Laenen, T. Boon, et al. Glycogen Synthase Kinase-3beta Phosphorylates Protein Tau and Rescues the Axonopathy in the Central Nervous System of Human Four-repeat Tau Transgenic Mice J. Biol. Chem., December 22, 2000; 275(52): 41340 - 41349. [Abstract] [Full Text] [PDF]
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Y. Huang, X. Q. Liu, T. Wyss-Coray, W. J. Brecht, D. A. Sanan, and R. W. Mahley Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons PNAS, July 17, 2001; 98(15): 8838 - 8843. [Abstract] [Full Text] [PDF]
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R. L. Raffai, L.-M. Dong, R. V. Farese Jr., and K. H. Weisgraber Introduction of human apolipoprotein E4 "domain interaction" into mouse apolipoprotein E PNAS, September 25, 2001; 98(20): 11587 - 11591. [Abstract] [Full Text] [PDF]
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, n =
3) and Thy1-ApoE4 transgenic
(
, n =
3) mice. Data were obtained by densitometric
analysis of immunoblots and normalized.
