Overexpression of Human Apolipoprotein B-100 ... - ACS Publications

To reveal the possible role of apoB-100 in neurodegeneration, we analyzed the serum lipoprotein and cerebral protein profiles, amyloid plaque formatio...
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Overexpression of Human Apolipoprotein B-100 Induces Severe Neurodegeneration in Transgenic Mice Erika Bereczki,†,# Ga´bor Berna´t,‡,#,¶ Tama´s Csont,§ Pe´ter Ferdinandy,§ Henning Scheich,‡ and Miklo ´ s Sa´ntha*,† Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary, Special Lab Non-Invasive Brain Imaging, Leibniz-Institute for Neurobiology, Brenneckestr. 6, D-39118 Magdeburg, Germany, and Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Do´m te´r 9. H-6720 Szeged, Hungary Received October 2, 2007

Recent studies showed correlation between increased serum apolipoprotein B-100 (apoB-100) level and Alzheimer’s disease. To reveal the possible role of apoB-100 in neurodegeneration, we analyzed the serum lipoprotein and cerebral protein profiles, amyloid plaque formation, apoptosis and brain morphology of transgenic mice overexpressing the human apoB-100 protein. Serum lipoprotein profile showed significant increase of the plasma triglyceride level, while no alteration in total cholesterol was detected. The antibody microarray experiment revealed upregulation of several cytoskeletal, neuronal proteins and proteins that belong to the mitogen activated protein kinase pathway, indicating active apoptosis in the brain. Histochemical experiments showed formation of amyloid plaques and extensive neuronal death. Biochemical changes severely affected brain morphology; a dramatic genotype-dependent enlargement of the third and lateral ventricles in the brain was detected. On the basis of earlier and present results, we conclude that overexpressed human apoB-100 protein significantly increases the level of serum lipids (triglyceride upon normal chow diet and cholesterol on cholesterol-rich diet) which leads to cerebrovascular lesions and subsequently induces apoptosis and neurodegeneration. Keywords: Apolipoprotein B-100 • cerebral protein profile • transgenic mice • magnetic resonance imaging • neurodegenerative disorders • ventricle enlargement

Introduction Apolipoprotein B-100 (apoB-100) is a large, 512 kDa glycoprotein that circulates in the plasma as the major protein component of low density lipoprotein (LDL). It is synthesized in the liver and is required for the formation and secretion of triglyceride-rich very low density lipoproteins for plasma cholesterol transport.1 Recent studies show that the human neurodegenerative disorder, Alzheimer’s disease (AD), is accompanied by elevated apolipoprotein B concentration in the serum2,3 and high serum level of apoB-100 modulates cerebral Aβ deposition in vivo.4 AD is characterized by progressive memory loss and cognitive impairment accompanied by neural degeneration, formation of amyloid plaques. Abnormal accumulation of apolipoprotein and cholesterol in the brain of AD patients has been detected as core components of mature * Corresponding author: Miklo´s Sa´ntha DVM, Ph.D., Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary. Tel: 36.62.599-651. Fax: 36.62.433-506. E-mail: [email protected]. † Hungarian Academy of Sciences. ‡ Leibniz-Institute for Neurobiology. # These authors contributed equally to this work. ¶ Present address: Lehrstuhl fu ¨r Biochemie der Pflanzen, Ruhr-Universita¨t Bochum, D-44801 Bochum, Germany. § University of Szeged.

2246 Journal of Proteome Research 2008, 7, 2246–2252 Published on Web 05/13/2008

amyloid plaques.5 Cholesterol content in neuronal membranes contributes to the maintenance of neuronal plasticity and might directly modulate the rate of amyloid precursor protein (APP) processing.6 A disturbance or imbalance in the sterol metabolism has been found in AD and vascular dementia, evidenced by an increase in the cholesterol breakdown product 24Shydroxycholesterol.7 It was recently demonstrated that elevation of plasma triglyceride level precedes amyloid deposition in Alzheimer’s disease model mice.8 Although apolipoprotein and cholesterol research was previously mainly focusing on cardiovascular diseases, latest findings indicate that apoB-100 might be involved in the development of neurodegenerative processes. To clarify whether apoB-100 has a role indeed in the process of neurodegeneration, we used cerebral protein profiling of transgenic mice overexpressing human apoB-100 generated earlier in our laboratory.9,10 To support our hypothesis, we analyzed the serum lipoprotein profile, apoptosis, amyloid plaque formation, and brain morphology of apoB-100 transgenic mice.

Materials and Methods Animals. Transgenic mice overexpressing the human apolipoprotein B-100 were generated in our laboratory as described earlier.9,10 The transgenic construct contained the entire 43 kb 10.1021/pr7006329 CCC: $40.75

 2008 American Chemical Society

Neurodegeneration in ApoB-100 Transgenic Mice human apoB gene, a 19 kb of the 5′ and a 14 kb of the 3′ flanking genomic sequences.11 The expression of human apoB100 transgene was detected by using Western blot analysis and immunohistochemistry. The best expressing transgenic line (485) was selected for further studies. This line was backcrossed with C57B6 mice three times in order to obtain more homogeneous genetic background. Mouse strain B6C3-Tg(APPSwe × PSEN1dE9) 85Dbo/J, a validated model of Alzheimer’s disease, was purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were kept on regular rodent chow diet and homozygous mice (human apoB-100 +/+) were investigated at the age of 6-7 months. Brain imaging experiments were performed on 6-7 month old animals that weighed 18-23 g (females) or 25-31 g (males). A total number of 10 homozygous, 6 heterozygous and 10 wild-type mice were used for the MR studies. The experimental protocol was approved by the local animal care ethical committee (license No. XIX/05004/ 001/2005). Determination of Serum Lipids. Plasma total cholesterol and triglyceride levels were measured in triplicate, using commercially available colorimetric assay kits, adapted to 96well plates (Diagnosticum Ltd., Budapest, Hungary). Accuracy of the assays was monitored by using Standard Lipid Controls (Sentinel, Milan, Italy). Values are expressed as millimoles per liter (mmol/L). Antibody Array Studies. Panorama AB Microarray Cell Signaling Kit (Sigma, CSAA1) was used to perform antibody array studies, according to the manufacturer’s instructions. This array contains 224 antibodies against proteins involved in apoptosis, cell cycle, cellular stress, signal transduction, as well as to nuclear, cytoskeletal and neuronal proteins. Proteins were extracted from freshly removed brain of 7 month-old transgenic and age-matched control mice according to the manufacturer’s instructions (Sigma) in buffer supplied with the kit. One milliliter (1 mg/mL) from this extract was labeled with Cy3 and Cy5 fluorescent dyes. The unincorporated dye was removed from the labeled samples using SigmaSpin columns. After mixing with 5 mL of incubation buffer (supplied in the kit), labeled samples were incubated on the arrays for 45 min and washed then four times for 5 min with washing buffer (supplied with the kit). The incubation and the washing procedures were done in the dark at room temperature on a shaker (30 rpm). Arrays were scanned using ScanArray LITE Microarray Analysis System (GSI Lumonics, Billerica, MA). Fluorescence intensities were normalized to that of the reference proteins and to the summed fluorescence intensities. Normalization by dye swapping was also done. Western Blot Analysis. Whole serum and total protein from mouse brain and liver samples were separated on 10% SDSPAGE precast gels (Cambrex), and transferred electrophoretically to Hybond P (Amersham) nitrocellulose membrane. Membranes were blocked for 1 h with 2% (w/v) ECL Advance Blocking Agent (Amersham) in PBST (1% phosphate buffered saline, pH 7.4; 0.1% Tween 20 v/v) at room temperature. After a brief washing in PBST, nitrocellulose membranes were probed overnight at 4 °C with different antibodies, namely, goat polyclonal anti-apoB-100 (1:1000, Chemicon), monoclonal antiAPP C-terminal (1:2000, Chemicon), rabbit anti-Pyk2 (1:3000, Sigma), rabbit anti-nNOS (1:2000, Chemicon), monoclonal antiiNOS (1:2000, Chemicon), monoclonal anti-PKC (1:2000, Chemicon), and monoclonal Hsp70 (1:5000, Chemicon) and then washed again in PBST. All incubations were performed on an orbital shaker. After 1 h incubation with the correspondent

research articles secondary antibody, ECL Advance Western Blotting Detection kit (Amersham) was used for detection, according to the manufacturer’s instructions. The anti-human apoB-100 polyclonal antibody recognized the endogenous mouse apoB-100 protein. Congo-Red Staining. Snap-frozen, 10 µm coronal and horizontal brain cryosections were stained with 10% Mayer’s hematoxylin for 10 min, and briefly washed with tap water. The slides were then differentiated in ethanol and washed with tap water for 5 min. Cryosections were stained with ∼50 mL of Congo-red dye dissolved in 80% ethanol saturated with NaCl, and 0.5 mL of 1% (w/v) NaOH for 20 min. After a brief rinse and dehydration step in 100% ethanol, slides were mounted and visualized under microscope. TUNEL Assay. Apoptosis-induced nuclear DNA fragmentation was detected using BD ApoAlert DNA Fragmentation Assay Kit (Clontech, Montain View, CA) according to the manufacturer’s protocol. Briefly, freshly frozen brain cryosections (10 µm) were digested with proteinase K (20 µg/mL) and washed in PBS. After fixation (4% formaldehyde/PBS) and subsequent equilibration, U TdT transferase (in incubation buffer) was added to the sections. Tailing reaction was performed for 60 min in a humidified incubator at 37 °C in the dark. Reaction was terminated by washing sections in 2× SSC solution for 15 min. After repeated washing in PBS (2 × 5 min), nuclei were stained with propidium iodide (Sigma). Apoptotic cells exhibiting nuclear green fluorescence signal were visualized under microscope using a 520 nm fluorescein filter set. TUNEL positive cells with diameter greater than 3 µm were counted and quantified by visually examining serial horizontal and coronal brain sections. Immunohistochemistry. Immunohistochemical analyses were performed on acetone-fixed brain cryosections (10 µm) using rabbit anti-β-Amyloid antibody (Chemicon) recognizing β-amyloid 1-40/42. To improve antibody penetration into the tissues, sections were treated with 0.5 mg/mL collagenase type II (Sigma) in a buffer containing 0.25 M NaCl, 50 mM TrisHCl, pH 7.4, and 1 mM EDTA at 37 °C for 30 min. To reduce nonspecific binding, sections were incubated in 10% normal donkey serum in TBS for 1 h. To visualize the nucleus, DAPI staining (Sigma) was used. Subsequently, fluorescein-conjugated donkey anti-rabbit-Cy3 (Jackson Immunoresearch) secondary antibody was used. Cryosections of corresponding brain regions of age-matched wild-type mice were used as negative controls, and corresponding APP(Swe)×Pse1 brain cryosections were used as positive controls. Animal Preparation for MR Imaging. Anatomical as well as manganese-enhanced magnetic resonance images (MEMRI) were recorded (on different days) on each animal. MEMRI was applied to enhance the contrast between the cerebrospinal fluid (brain ventricles) and the brain tissues, this being a prerequisite for accurate ventricle size quantification. Two days before the MEMRI measurements, mice were injected subcutaneously with 10 µL of MnCl2/g body weight from a 100 mM stock solution of MnCl2. During the MRI measurements and for the MnCl2 administration, animals were anesthetized with 1.0-1.5% isoflurane in 7:3 (v/v) N2O/O2 mixture. To avoid the drying out of the animal’s nose, the gas flow was humidified by bubbling through distilled water. Respiration was monitored by LabVIEW (National Instruments, Austin, TX). Isoflurane concentration was adjusted manually according to the observed respiratory frequency in order to maintain 90-100 breaths per minute. The anesthetized mice were immobilized in prone position by using Journal of Proteome Research • Vol. 7, No. 6, 2008 2247

research articles a home-built setup with head holder (through which anesthetic gas was flowing) and bite bar to reduce motion artifacts and to maintain anesthesia. Body temperature was maintained at 37 °C by laying the animals on a plastic plate with water circulation. Animals were sacrificed after the MEMRI measurements by overdosing of isoflurane. Magnetic Resonance Imaging. Morphological differences in the central nervous system were detected and quantified by MR experiments performed on a Bruker Biospec 47/20 MRI scanner (Bruker BioSpin, Ettlingen, Germany) operating at 4.7 T (with inner diameter of 20 cm). The instrument was equipped with an actively shielded BGA 12 (200 mT/m) gradient system and controlled by ParaVision operating software (version 3.0.2, Bruker). RF excitation and signal reception were accomplished with the use of a 25 mm (i.d.) Litzcage coil system (DotyScientific, Columbia, SC). Anatomical images (eight horizontal T2-weighed images) were obtained simultaneously using a rapid acquisition relaxation enhanced sequence12 with the following parameters: repetition time (TR) ) 2000 ms; echo time (TE) ) 15.00 ms; slice thickness ) 800 µm; field of view (FOV) ) 3 × 3 cm; matrix dimensions ) 256 × 256; rare factor ) 8; number of averages ) 8. The total scanning time was 9 min. Continuous high-resolution, three-dimensional data sets were acquired by using a T1-weighing Modified Driven Equilibrium Fourier Transform imaging sequence. The imaging parameters were the followings: TR ) 21.18 ms; TE ) 4.00 ms; flip angle ) 15°; FOV ) 2 × 2 × 2 cm; matrix dimensions ) 256 × 256 × 64; number of averages ) 10. The total scanning time was 1.5 h. Data Processing and Statistical Analysis. The total brain volume and the cavity size of the ventricular system of each individual animal were established using the public domain Java image processing program ImageJ (http://rsb.info.nih.gov/ ij/). Number of voxels belonging either to the brain tissue or to the ventricles was determined manually outlining section by section (≈50 axial sections/brain); the total volumes were obtained by multiplying the number of voxels involved and the size of one individual voxel (≈78 × 78 × 312.5 µm). The ventricle-to-brain ratios were quantified and expressed as mean ( standard deviation; statistical significance of differences between the control, hetero-, and homozygous groups were evaluated using one-way ANOVA for independent samples. The level of statistical confidence was set at p < 0.05.

Results Generation and Characterization of ApoB-100 Transgenic Mice. Generation of transgenic mice overexpressing the human apoB-100 gene was described earlier.9,10 First, expression of human apoB-100 protein in different organs of transgenic mice was tested. Dose-dependent expression of the transgene was detected in the liver and serum of apoB-100 transgenic line 485 using semiquantitative Western blot analysis (Figure 1a,b). Transgene expression was also found in the heart, aorta and eyes of transgenic mice using Western analysis, whereas no apoB-100 immunoreactivity was observed in the brain using immunohistochemistry (data not shown). Then, plasma total cholesterol and triglyceride levels of transgenic offspring were determined. The total cholesterol level was not changed in transgenic mice compared to wild-type controls, whereas the triglyceride level increased significantly (0.72 ( 0.25 mmol/L 2248

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Figure 1. (a) Detection of ApoB-100 protein from the liver (a) and serum (b) of wild-type (Wt), heterozygous (Tg+/-) and homozygous (Tg+/+) transgenic mice using Western blot analysis (Ponceau staining is shown as loading control). (c) Serum total cholesterol and triglyceride levels in wild-type (Wt) and apoB100 transgenic mice (Tg+/+) fed normal chow diet. Data represent the means ( standard deviations (n ) 7 in each group), ** p < 0.0099. (d) Western blot analysis of APP, Pyk2, nNOS, iNOS, PKC and Hsp70 proteins derived from wild-type control (Wt) and homozygous apoB-100 transgenic (Tg+/+) brain samples. Mouse β-actin antibody was used as an internal control.

in wild-type versus 1.47 ( 0.57 mmol/L in apoB-100 transgenic mice; p < 0.0099) (Figure 1c). Extended Apoptosis and Appearance of Amyloid Plaques in the Brain of Apo-B Transgenic Mice. Activation of apoptotic pathway was investigated by cerebral protein profiling of apoB100 transgenic mice using Panorama AB Microarray Cell Signaling Kit. We found an approximately 2-fold increase in the level of signaling proteins, such as the nonreceptor tyrosine kinase Pyk2, MAPK (mitogen activated protein kinase) activated diphosphothreonine, p38MAPK, RAF1 (rapidly accelerated fibrosarcoma kinase), FAK (focal adhesion kinase), nNOS (nitric oxide synthase) and iNOS (Table 1). Additionally, remarkable decrease in the level of PKC β (protein kinase Cβ2) expression was observed (Table 1). Altered levels of Pyk2, nNOS, iNOS and PKC proteins were confirmed by Western blot analysis (Figure 1d). We also found elevated expression of certain neuronal proteins such as S-100 β, amyloid precursor protein, glutamine synthetase (GS), syntaxin and cellular stress protein, Hsp70, in the brain of apoB-100 transgenic mice (Table 1). Increased level of APP and Hsp70 proteins was also confirmed by Western blot analysis (Figure 1d). Cerebral protein profiling of apoB100 trangenics revealed an approximately 2-fold increase in the level of cytoskeletal proteins, such as γ-tubulin and OP18/ stathmin (Table 1). Activation of the apoptotic pathway in the brain of apoB100 transgenic mice was further studied using TUNEL assay. When compared to wild-type controls, extensive neuronal death was detected in the hippocampus, cerebral cortex and hypothalamusofapoB-100transgenicaswellasinAPP(Swe)×Pse1 double transgenic mice, a validated mouse model of AD, used

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Table 1. List of Antibodies from Panorama AB Microarray Showing Altered Protein Expression in ApoB-100 transgenic Brain spot

antibody name

sigma acc. no.

area

fold overexpression

5.3Ccd 5.4Aab 5.1Dab 5.2Dab 6.1Bcd 6.3Ccd 6.1Cab 6.1Acd 7.2Dab 7.3Cab 8.1Dab 8.3Dcd 8.3Bcd 7.2Bab 3.2Dab

γ-Tubulin OP18/Stathmin iNOS nNOS Glutamine Synthethase Syntaxin S-100 β Amyloid Precursor Protein C-terminal MAP Kinase activated diphosphothreonine p38 MAPK activated Pyk2 RAF1 PKC β FAK Hsp70

T3559 O0138 N7782 N7155 G2781 S0664 S2532 A8717 M8159 M8177 P3902 R5773 P3203 F9301 H5147

Cytoskeleton Cytoskeleton Signal transduction Signal transduction Neurobiology Neurobiology Neurobiology Neurobiology Signal Transduction Signal Transduction Signal Transduction Signal Transduction Signal Transduction Signal Transduction Cell stress

1.91 1.96 1.92 1.93 2.25 2.55 2.18 1.91 2.13 1.98 2.15 2.46 0.15 1.80 2.06

as positive control in this experiment (Figure 2). Stained apoptotic neurons were counted and statistical analysis showed significant differences in the number of apoptotic neurons in hippocampal and cerebral cortical regions of wild-type, heterozygous ApoB-/+, homozygous ApoB+/+, and APP(Swe)×Pse1 transgenic mice (Figure 2e). Elevated APP level and extensive neuronal death indicate the possible formation of amyloid plaques in the brain of apoB100 transgenic mice. To detect aggregated β-amyloid deposits, Congo-red staining and β-amyloid immunostaining was used. Amyloid plaques were detected with preponderance in the hippocampal, cortical and hypothalamic regions of ApoB-100 and APPSwe×PSEN1 transgenic mice (Figure 3b-d and f-h) while no or very little staining was detected in brain cryosections of wild-type mice (Figure 3a,e). Neuroimaging Confirms Severe Neurodegeneration. We hypothesized that the extensive neuronal death should affect brain morphology. This was investigated using magnetic

resonance imaging. Large increase in the cavity size of the lateral and dorsal ventricles and a moderate enlargement in the aqueduct (fourth ventricle) were detected in the brain of heterozygous transgenic animals (Figure 4a). Moreover, the enlargement of the ventricular system was more pronounced in homozygote than in heterozygote transgenic mice (Figure 4a). No volume changes in other brain compartments were found by quantitative segmentation of the individual images (not shown). There was no significant difference in the total brain volume of the different groups (454 ( 16, 454 ( 16, 476 ( 39 mm3 volume in the control, hetero-, and homozygous group, respectively). Constructing complete 3D structures of the ventricular system from manganese-enhanced image-series of about 50 thin (312.5 µm) axial layers reveals more details (Figure 4b) and refines the view obtained by two-dimensional, anatomical images with larger (800 µm) thickness (Figure 4b). Statistical analysis shows that in the control group the relative volume of the third ventricle (including lateral ventricles) and

Figure 2. Staining of apoptotic cells on brain slices of wild type (a), APP(Swe)×Pse1 (b) and apoB-100 transgenic mice (c and d) using TUNEL assay. Apoptosis-induced nuclear DNA fragmentation was detected in the hippocampal, and cerebral cortical regions (white arrows) of horizontal brain sections of apoB-100 transgenic mice (c and d) as well as of APP(Swe)×Pse1 mice (b). APP(Swe)×Pse1 transgenic mice are validated model of Alzheimer’s disease and were used as a positive control throughout this experiment. TUNEL positive cells were counted separately in the cerebral cortex and hippocampus of wild-type, heterozygous apoB-/+ and homozygous apoB+/+ and APP (Swe)×Pse1 transgenic mice (e). Error bars and significance levels (*p < 0.05) are indicated. (White scale bar represents 25 µm.) Journal of Proteome Research • Vol. 7, No. 6, 2008 2249

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Figure 3. Congo-red staining of brain slices of wild-type (a), APP(Swe)×Pse1 (b) and apoB-100 transgenic mice (c and d). Deposits of aggregated β-amyloid are indicated with black arrows (black scale bar represents 30 µm). β-Amyloid immunostaining of wild-type (e), APP(Swe)×Pse1 (f) and apoB-100 transgenic (g and h) brain cryosections. White arrowheads indicate β-amyloid aggregations (white scale bar represents 25 µm).

the fourth ventricle (including the aqueduct) was 0.41 ( 0.12% and 0.30 ( 0.06%, respectively. This corresponds to about 1.8 and 1.4 µL of cerebrospinal fluid (3.2 µL total) in the brain. In the human apoB-100 overexpressing animals, the enlargement of ventricles was proportional to the increased human apoB100 level. Mean values of the third/lateral ventricle volume were 4.9 and 8.7 µL in the hetero- and homozygous animals, respectively (Figure 4c). When apoB transgenic mice were fed cholesterol-rich diet, the serum total cholesterol level increased significantly, while the level of triglyceride remained as low as in control, wildtype mice.10 Morphologically, similar enlargement of the third and fourth ventricles was observed in the brain of apoB-100 mice fed with cholesterol-rich diet using MRI and MEMRI as in transgenic mice fed normal chow (data not shown).

Discussion In this study, we showed that overexpression of human apoB-100 in transgenic mice leads to significant increase of the plasma triglyceride level, changes in the cerebral protein profile, and triggers apoptosis in the brain. Activation of the apoptotic pathway begins with the elevation of Pyk2, which acts as an upstream regulator of p38MAPK signaling pathway.13 Parallel downregulation of PKC β also indicates the active state of the apoptotic cascade. Similarly to our findings, hyperactivation of the apoptotic signaling pathway was observed earlier in Alzheimer’s disease.14,15 Besides extended apoptosis, other signaling pathways providing increased survival to neurons were also activated as indicated by the elevated level of FAK and RAF1. Supporting our hypothesis, we detected extensive neuronal death in the hippocampus, cerebral cortex and hypothalamic regions of brain sections of apoB-100 transgenic mice using TUNEL assay. Similar result was obtained for Alzheimer’s disease model, APP(Swe)×PS1 double transgenic mice. Interestingly, parallel to the activation of signaling pathways, the level of brain injury indicating proteins such as S-100 β, nNOS, glutamine synthetase (GS) and Hsp70 was also increased indicating a certain impact of apoB-100 in neurodegeneration. Aberrant production of S-100 β which stimulates the expression 2250

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of NOS via p38 MAPK activation16 has been already reported in several neurodegenerative diseases, including Alzheimer’s disease.17 It was also shown that accumulation of nitric oxide (NO), synthesized by NOS enzyme family, mediates neurotoxic effect and triggers tau hyperphosphorylation in hippocampal neurons.18 Overexpression of APP751 protein in APP23 transgenic mice induced glial iNOS and eNOS expressions and resulted in amyloid plaque formation.19 We found increased APP level in the brain of transgenic mice overexpressing apoB100. Earlier, we have shown that the level of protein kinase C, a known regulator of R-secretory proteolytic processing of the APP and β-secretase, is not altered;9 therefore, cerebral APP might accumulate and can go through aberrant cleavage by γ-secretases resulting in accumulation of toxic Aβ (1-42) peptides. Soluble Aβ oligomers include spherical particles and curvilinear structures called “protofibrils”20 and they are most likely the precursors of Aβ fibrils and amyloid plaques. Using Congo-red staining, we were able to detect amyloid plaque formation with preponderance in the hippocampus, cerebral cortex and hypothalamic region of brain slices of apoB-100 transgenic mice. β-Amyloid immunostaining detected increased number of amyloid aggregates in the cerebral cortex and hippocampus of transgenic mice compared to wild-type mice. Presence of amyloid plaques is very characteristic for neurodegenerative disorders, such as Alzheimer’s disease; therefore, we think that apoB-100 transgenic mice can be a suitable model of this disease. The loss of neuronal membrane phospholipids and fatty acids has been hypothesized to be an early metabolic event in the formation of amyloid plaques.21 Certainly, further characterization of cerebral lipid metabolism, determination of the membrane content and distribution of docosahexaenoic acid (DHA) and other omega-3 long chain poly unsaturated fatty acids (LC-PUFA) as well as monitoring the expression level of apoA, ApoE and LDL receptor are necessary. It is known that excess of glutamate is neurotoxic and induces apoptosis of neural cells.22 On the other hand, glutamine synthetase confers neuroprotection via elimination of neurotoxic glutamate from neurons.23 Increased amount of glutamine synthetase enzyme was found in the prefrontal cortex of Alzheimer’s disease patients.24 As elevated level of GS can be

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Figure 4. MRI images of wild-type (-/-), hetero- (+/-) and homozygous (+/+) apoB-100 transgenic mice. (a) Horizontal T2weighed MR images at the level of dorsal hippocampus. For clarity, inverse of the original grayscale was used. (b) 3D image of the ventricular system constructed from a series of individual coronal T1-weighted slices. The ventricular system is highlighted in gray and projected as a front-view. (c) Volumetric analysis of the cerebroventricular system in apoB-100 transgenic mice. White, gray and black columns represent the volume of the fourth ventricle with the aqueduct, the third ventricle with the lateral ventricles and the total volume, respectively. Error bars and significance levels (* p < 0.05 and ** p < 0.001) are indicated.

detected in the cerebrospinal fluid of Alzheimer’s disease patients, its application as a diagnostic biochemical marker for AD has been proposed.25 We also found an increase in the levels of neural protein, syntaxin, and cytoskeletal protein, stathmin, in the brain of apoB-100 transgenic mice. Syntaxin 1A, a presynaptic plasma membrane protein, can directly bind to presenilin-1; therefore, its overexpression can modulate γ-secretase activity26 that plays a central role in the neuropathogenesis of Alzheimer’s disease. It was speculated previously that Hsp70 might exert a role in the enhancement of protein degradation,27 but recently, it is considered as a mediator of neuroprotective response by mitigating the degenerative effect conferred by R-synuclein.28 Stathmin can directly bind to tubulin and both in its native and phosphorylated forms destabilize assembled microtubules, thus, disrupting microtubular structure.29 Similar increase in stathmin immunoreactivity was reported earlier in hippocampal neurons of AD patients.18

Morphological investigations of the brain of apoB-100 transgeic mice confirmed extensive neuronal apoptosis and severe neurodegeneration. Moreover, the enlargement of the lateral ventricles showed correlation with the transgene copy number indicating the primary role of apoB-100 excess in neurodegeneration. Although we show here that overproduction of apoB-100 results in severe neurodegeneration, the exact mechanism of this process is not known. One plausible explanation is the elevated plasma triglyceride or cholesterol level. This hypothesis is in accordance with recent findings of Eiselein and coworkers, who showed that lipolytic products from triglyceriderich lipoproteins increase endothelial permeability, perturb the expression of junctional proteins and induce apoptosis in vitro.30 When the serum lipoprotein content of three transgenic AD mouse models was analyzed, it was demonstrated that increased plasma triglyceride level precedes amyloid deposition in the brain of AD model mice, whereas the total cholesterol level remains unchanged during neurodegeneration.8 After 18 weeks of cholesterol-rich diet, the level of total cholesterol and LDL cholesterol significantly increased in the transgenic group of mice, while the triglyceride level remained low, comparable to the wild-type animals.10 However, the lateral ventricles and aqueduct were enlarged in the same extent as in transgenic animals fed normal chow diet, indicating that not only elevetad serum triglycerides level but hypercholesteremia as well can induce the same effect. Our previous and present results show that overexpression of apoB-100 is accompanied by chronic hypertriglyceridemia (normal chow diet) or hypercholesteremia (cholesterol-rich diet) and either forms of hyperlipidemia can lead to alteration of cerebral protein profile and induce neuronal apoptosis and neurodegeneration. Recent studies indicate a link between age-related vascular lesions and neurodegenerative diseases.31–34 de Leeuw and his co-workers showed that periventricular white matter lesions (which are associated with dementia) were increased in those patients who had aortic atherosclerosis at middle age.35 Newman et al. found that carotid artery wall thickness and anklearm index (an indicator of extent of peripheral arterial disease) were associated with and increased risk of AD.36 Our results support these findings and underline the crucial role of hyperlipidemia in the development of vascular pathology and subsequent neuronal apoptosis and neurodegeneration. Previously, we have shown that apoB-100 overexpressing hypertriglyceridemic mice show elevated serum level of malondialdehyde (MDA), a marker of systemic lipid peroxidation.10 ApoB100 transgenic mice fed cholesterol-rich diet showed elevated level of serum low density lipoprotein (LDL) and developed atherosclerotic plaques by the age of 7 months.10 They also showed an increase in the level of serum MDA, myocardial NADPH oxidase, superoxide, and nitrotyrosine.10 Chronic effects of hyperlipidemia (hypertriglyceridemia or hypercholesterolemia) most probably affect not only the cardiovascular, but the cerebrovascular system as well, leading to progressive arterial lesions and subsequently neuronal apoptosis.

Conclusion In conclusion, our present study revealed that overexpression of human apoB-100 in transgenic mice leads to multiple and profound alterations in the cerebral protein profile and induce neuronal apoptosis and neurodegeneration. Obviously, further experiments such as time-course analysis of neurodegeneration Journal of Proteome Research • Vol. 7, No. 6, 2008 2251

research articles and behavioral assays confirming learning deficits and memoryloss are necessary to reveal the exact mechanism of apoB-100 triggered neurodegeneration. If further results confirm current observations, then these mice would become a valuable novel model of human neurodegenerative disorders.

Acknowledgment. The authors are grateful to Dr. Dirk Montag and Dr. Ja´nos Z. Kelemen for their help in animal accommodation and microarray data analysis, respectively. We thank Drs. Frank Angenstein and Heiko G. Niessen for stimulating discussions and to Dr. Istva´n Ando´ and Rui M. M. Branca for critical reading of the manuscript. The financial support from the EU Program “Improving Human Research Potential” (HPMD-CT-2001-00068 to G.B.) and the National Office for Research and Technology (RET-08/2004 to M.S.) are gratefully acknowledged. References (1) Chan, L. Apolipoprotein-B, the major protein component of triglyceride-rich and low-density lipoproteins. J. Biol. Chem. 1992, 267, 25621–25624. (2) Caramelli, P.; Nitrini, R.; Maranhao, R.; Lourenco, A. C. G.; Damasceno, M. C.; Vinagre, C. Caramelli, B. Increased apolipoprotein B serum concentration in Alzheimer’s disease. Acta Neurol. Scand. 1999, 100, 61–63. (3) Sabbagh, M.; Zahiri, H. R.; Ceimo, J.; Cooper, K.; Gaul, W.; Connor, D.; Sparks, D. L. Is there a characteristic lipid profile in Alzheimer’s disease. J. Alzheimer Dis. 2004, 6, 585–589. (4) Kuo, Y. M.; Emmerling, M. R.; Bisgaier, C. L.; Essenburg, A. D.; Lampert, H. C.; Drumm, D.; Roher, A. E. Elevated low-density lipoprotein in Alzheimer’s disease correlates with brain A beta 142 levels. Biochem. Biophys. Res. Commun. 1998, 252, 711–715. (5) Puglielli, L.; Tanz, R. E.; Kovacs, D. M. Alzheimer’s disease: the cholesterol connection. Nat. Neurosci. 2003, 6, 345–351. (6) Ehehalt, R.; Keller, P.; Haass, C.; Thiele, C.; Simons, K. Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J. Cell. Biol. 2003, 160, 113–123. (7) Lutjohann, D.; Papassotiropoulos, A.; Bjorkhem, I.; Locatelli, S.; Bagli, M.; Oehring, R. D.; Schlegel, U.; Jessen, F. and others. Plasma 24S-hydroxycholesterol (cerebrosterol) is increased in Alzheimer and vascular demented patients. J. Lipid Res. 2000, 41, 195–198. (8) Burgess, B. L.; McIsaac, S. A.; Naus, K. E.; Chan, J. Y.; Tansley, G. H. K.; Yang, J.; Miao, F. D.; Ross, C. J. D. and others. Elevated plasma triglyceride levels precede amyloid deposition in Alzheimer’s disease mouse models with abundant A beta in plasma. Neurobiol. Dis. 2006, 24, 114–127. (9) Bjelik, A.; Bereczki, E.; Gonda, S.; Juhasz, A.; Rimanoczy, A.; Zana, M.; Csont, T.; Pakaski, M. and others. Human apoB overexpression and a high-cholesterol diet differently modify the brain APP metabolism in the transgenic mouse model of atherosclerosis. Neurochem. Int. 2006, 49, 393–400. (10) Csont, T.; Bereczki, E.; Bencsik, P.; Fodor, G.; Gorbe, A.; Zvara, A.; Csonka, C.; Puskas, L. G. and others. Hypercholesterolemia increases myocardial oxidative and nitrosative stress thereby leading to cardiac dysfunction in apoB-100 transgenic mice. Cardiovasc. Res. 2007, 76, 100–109. (11) Callow, M. J.; Stoltzfus, L. J.; Lawn, R. M.; Rubin, E. M. Expression of human apolipoprotein-B and assembly of lipoprotein (A) in transgenic mice. Procl. Natl. Acad. Sci. U.S.A. 1994, 91, 2130–2134. (12) Hennig, J.; Nauerth, A.; Friedburg, H. RARE Imaging- a fast imaging method for clinical MR. Magn. Reson. Med. 1986, 3, 823–8339. (13) Tian, D. H.; Litvak, V.; Lev, S. Cerebral ischemia and seizures induce tyrosine phosphorylation of PYK2 in neurons and microglial cells. J. Neurosci. 2000, 20, 6478–6487. (14) Mattson, M. P. Apoptosis in neurodegenerative disorders. Nat. Rev. Mol. Cell. Biol. 2000, 1, 120–129. (15) Saez, T. E.; Pehar, M.; Vargas, M.; Barbeito, L.; Maccioni, R. B. Astrocytic nitric oxide triggers tau hyperphosphorylation in hippocampal neurons. In Vivo 2004, 18, 275–280. (16) Esposito, G.; De Filippis, D.; Cirillo, C.; Sarnelli, G.; Cuomo, R.; Iuvone, T. The astroglial-derived S100 beta protein stimulates the

2252

Journal of Proteome Research • Vol. 7, No. 6, 2008

Bereczki et al.

(17) (18) (19)

(20)

(21)

(22) (23)

(24)

(25)

(26) (27)

(28)

(29) (30)

(31) (32) (33) (34) (35)

(36)

expression of nitric oxide synthase in rodent macrophages through p38 MAP kinase activation. Life Sci. 2006, 78, 2707–2715. Van Eldik, L. J.; Griffin, W. S. S100[beta] expression in Alzheimer’s disease: Relation to neuropathology in brain regions. Biochim. Biophys. Acta 1994, 1223, 398–403. Saitoh, T.; Horsburgh, K.; Masliah, E. Hyperactivation of signaltransduction systems in Alzheimers-disease. Ann. N.Y. Acad. Sci. 2003, 695, 34–41. Luth, H. J.; Holzer, M.; Gartner, U.; Staufenbiel, M.; Arendt, T. Expression of endothelial and inducible NOS-isoforms is increased in Alzheimer’s disease, in APP23 transgenic mice and after experimental brain lesion in rat: evidence for an induction by amyloid pathology. Brain Res. 2001, 91, 57–67. Hartley, D. M.; Walsh, D. M.; Ye, C. P.; Diehl, T.; Vasquez, S.; Vassilev, P. M.; Teplow, D. B.; Selkoe, D. J. Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J. Neurosci. 1999, 19, 8876–8884. Pettegrew, J. W.; Panchalingam, K.; Moossy, J.; Martinez, J.; Rao, G.; Boller, F. Correlation of phosphorus-31 magnetic resonance spectroscopy and morphologic findings in Alzheimer’s disease. Arch. Neurol. 1988, 45, 1093–1096. Vardimon, L. Neuroprotection by glutamine synthetase. Isr. Med. Assoc. J. 2005, 2, 46–51. Burbaeva, G. S.; Boksha, I. S.; Tereshkina, E. B.; Savushkina, O. K.; Starodubtseva, L. I.; Turishcheva, M. S. Glutamate metabolizing enzymes in prefrontal cortex of Alzheimer’s disease patients. Neurochem. Res. 2005, 30, 1443–1451. Gunnersen, D.; Haley, B. Detection of glutamine-synthetase in the cerebrospinal-fluid of Alzheimer diseased patients- a potential diagnostic biochemical marker. Proc. Natl. Acad. Sci. U.S.A. 1999, 92, 11949–11953. Smith, S. K. F.; Anderson, H. A.; Yu, G.; Robertson, A. G. S.; Allen, S. J.; Tyler, S. J.; Naylor, R. L.; Mason, G. and others. Identification of syntaxin 1A as a novel binding protein for presenilin-1. Mol. Brain Res. 2000, 78, 100–107. Yu, Y. X.; Shen, L.; Xia, P.; Tang, Y. W.; Bao, L.; Pei, G. Syntaxin 1A promotes the endocytic sorting of EAAC1 leading to inhibition of glutamate transport. J. Cell. Sci. 2006, 119, 3776–3787. Terlecky, S. R.; Chiang, H. L.; Olson, T. S.; Dice, J. F Protein and peptide binding and stimulation of invitro lysosomal proteolysis by the 73 kDa heat-shock cognate protein. J. Biol. Chem. 1992, 267, 9202–9209. Yu, F.; Xu, H.; Zhuo, M.; Sun, L.; Dong, A.; Liu, X. Impairment of redox state and dopamine level induced by [alpha]-synuclein aggregation and the prevention effect of hsp70. Biochem. Biophys. Res. Commun. 2005, 331, 278–284. Moreno, F. J.; Avila, J. Phosphorylation of stathmin modulates its function as a microtubule depolymerizing factor. Mol. Cell. Biochem. 1998, 183, 201–209. Eiselein, L.; Wilson, D.; Lame, M.; Rutledge, J. C. Lipolysis products from triglyceride rich lipoproteins increase endothelial permeability, perturb Zonula Occludens-1, F-Actin, and induce apoptosis. Am. J. Physiol.: Heart Circ. Physiol. 2007, 292, H2745–H2753. Hamel, E.; Nicolakakis, N.; Aboulkassim, T.; Ongali, B.; Tong, X. K. Oxidative stress and cerebrovascular dysfunction in mouse models of Alzheimer’s disease. Exp. Physiol. 2008, 93, 116–120. Kalaria, R. N. Linking cerebrovascular defense mechanism in brain ageing and Alzheimer’s disease. Neurobiol. Aging 2008, in press. Launer, L. J.; Petrovitch, H.; Ross, G. W.; Markesbery, W.; White, L. R. AD brain pathology: Vascular origins? Results from the HAAS autopsy study. Neurobiol. Aging 2007, in press. Stampfer, M. J. Cardiovascular disease and Alzheimer’s disease: common links. J. Intern. Med. 2006, 260, 211–223. de Leeuw, F. E.; De Groot, J. C.; Oudkerk, M.; Witteman, J. C.; Hofman, A.; van Gijn, J.; Breteler, M. M. Aortic atherosclerosis at middle age predicts cerebral white matter lesions in the elderly. Stroke 2000, 31, 425–429. Newman, A. B.; Fitzpatrick, A. L.; Lopez, O.; Jackson, S.; Lyketsos, C.; Jagust, W.; Ives, D.; Dekosky, S. T.; Kuller, L. H. Dementia and Alzheimer’s disease incidence in relationship to cardiovascular disease in the Cardiovascular Health Study cohort. J. Am. Geriatr. Soc. 2005, 53, 1101–1107.

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