| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
,1
* Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya, Japan;
Division of Clinical Science and Neuropsychopharmacology in Clinical Pharmacy Practice, Management and Research, Faculty of Pharmacy, Meijo University, Nagoya, Japan; and
Department of Vascular Dementia Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Morioka, Obu, Japan
2Correspondence: Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8560, Japan. E-mail: tnabeshi{at}med.nagoya-u.ac.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: Aß-peptide • cognitive dysfunction
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. The accumulation of amyloid ß-peptide (Aß) is proposed to be a trigger of the decades-long pathological cascade leading to the development of AD (2)
, since gene mutations of amyloid precursor protein (APP) (3
, 4)
and presenilin (PS) (5)
in patients with early-onset familial AD enhance the processing of APP to form Aß, resulting in an accelerated accumulation of Aß.
Current drug therapy for AD aims at slowing cognitive decline and ameliorating the affective and behavioral symptoms associated with the disease’s progression. However, these drugs provide limited symptomatic treatment without targeting the underlining cause of AD. Based on the Aß hypothesis, a variety of immunotherapies designed to remove Aß are being developed. The first of these treatments—active immunization with an intramuscular injection of preaggregated Aß42—prevented the development of Aß plaques in PDAPP mice (6)
. This active immunotherapy not only reduced Aß levels in the brain, but also ameliorated cognitive dysfunction in two other strains of APP transgenic mice (7
, 8)
. Passive immunotherapy with a systemically administered anti-Aß antibody also induced a reduction in the amount of Aß in the brain (9)
and attenuated memory deficits in APP transgenic mice (10)
. In phase II clinical trials, immunotherapy with Aß (AN-1792) reduced numbers of Aß plaques in the neocortex (11)
and attenuated the decline of cognitive functions in AD patients who generate antibody against Aß deficits (12)
. However, complications (subacute meningoencephalitics) appeared in 6% of the patients (13)
. This meningoencephalitics is thought to be caused by autoimmune T-lymphocytes, because leptomeningeal infiltration by T-lymphocytes was observed (11)
.
Our previous studies have shown that oral administration of a recombinant adeno-associated viral vector carrying Aß cDNA (AAV/Aß) induces the expression of Aß in the epithelial cell layer of the intestine in Tg2576 mice, one of several strains of APP transgenic mice. AAV/Aß reduces the amount of Aß deposited in the brain of Tg2576 mice without causing lymphocytic infiltration in the brain, suggesting that immunotherapy with AAV/Aß is a useful and safe treatment for AD (14)
. However, there is no report on whether AAV/Aß attenuates cognitive and histological abnormalities in Tg2576 mice. The present study was designed to test the hypothesis that AAV/Aß improves cognitive and histological abnormalities in Tg2576 mice accompanied by a decrease in the accumulation of Aß, since soluble Aß oligomers (Aß*56) and insoluble Aß are also critically responsible for cognitive dysfunction in Tg2576 mice (15
, 16)
. We attempted to investigate the effect of AAV/Aß on 1) cognitive function, 2) microglial attraction and presynaptic degeneration, 3) the formation of Aß plaques, and the amount of insoluble Aß and soluble Aß oligomers, and 4) lymphocytic infiltration and microhemorrhage in Tg2576 mice.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Contraction of the adeno-associated viral vector
A recombinant adeno-associated viral (AAV) vector for the expression of Aß1–43 was constructed using plasmid DNA pTRUF2 (17)
. Primers were designed to amplify cytosolic Aß1–43 from APP695. These primers amplify the entire Aß sequence with an additional stop codon at the 3' end to ensure translation. Cytosolic Aß was amplified with the Aß1–43 forward primer 5'-GAT GCA GAA TTC CGA CAT GAC TCA GGA-3' and reverse primer 5'-GTC TTA AGT CGC TAT GAC AAC ACC GCC C-3' (with a 3' AflII site). The secreted form of Aß was made by linking the APP signal peptide (SP) to the Aß sequence. The SP adaptor was assembled by annealing the forward oligonucleotide and the reverse oligonucleotide (has a single 3' T residue) after incubation at 90°C for 3 min. Ligation of the SP adaptor (contains a single 3' T residue) and PCR-amplified Aß coding sequence (has 3'A overhangs) was followed by reamplification with the SP forward primer 5'-GGT CTA GAA TGC TGC CCG GTT TGG CAC-3' (contains a 5' XbaI site) and Aß1–43 reverse primer. The PCR-amplified SP and Aß sequence (3' AflII/blunt) was further ligated to the nonfunctional "stuffer" sequences of pBR322 (PvuII-SalI fragment) to achieve a genome size (4465 bp) adequate for efficient AAV packaging. The SP and Aß nonfunctional "stuffer" sequences were cloned into the vector pTRUF2 through the XbaI/SalI restriction site.
Production of recombinant AAV
Human embryonic kidney (HEK) 293 cells were cotransfected with SP-Aß pTRUF2 and the plasmids pXX2 and pXX6 as described elsewhere (18)
. Recombinant AAV titers were in the range of 1 x 1013 to 2 x 1013 viral genomes per milliliter.
AAV/control, which expresses GFP, was produced as described before (17)
; recombinant AAV/Aß titers were also in the range of 1 x 1013 to 2 x 1013 viral genomes per milliliter.
Administration of the vector to mice
AAV/Aß or AAV/control was diluted with PBS to give 5 x 1011 genomes in a final volume of 0.1 ml and was orally administered once to mice at the age of 10 months.
Behavioral analysis
Experimental design
At the age of 6 and 10 months, mice (non-tg mice, n=28; Tg2576 mice; n=28) were subjected to behavioral tests. After the behavioral test at the age of 10 months, mice were divided into two groups to be treated with AAV/control or AAV/Aß. There is no difference between the two groups in behavioral scores at the age of 6 and 10 months. A previous report shows that treatment of AAV/Aß at the age of 10 months reduces the amount of Aß deposited in the brain of Tg2576 mice at the age of 12–13 months (14)
. Thus, to evaluate the effect of vaccine treatment, each group (AAV/control-treated non-tg mice, n=14; AAV/Aß-treated non-tg mice, n=14; AAV/control-treated Tg2576 mice, n=14; AAV/Aß-treated Tg2576 mice, n=14) was subjected to behavioral tests at 13 months of age.
Novel object recognition test
The novel object recognition test was performed in mice at the age of 6, 10, and 13 months according to a previous report (19)
, with minor modifications. The test procedure consisted of three sessions: habituation, training, and retention. Each mouse was individually habituated to the box (30x30x35 high cm), with 10 min of exploration in the absence of objects for 3 days (habituation session). During the training session, two objects were placed in the back corner of the box. A mouse was then placed midway at the front of the box and the total time spent exploring the two objects was recorded for 10 min. During the retention session, animals were placed back into the same box 24 h after the training session, in which one of the familiar objects used during training was replaced with a novel object. The animals were then allowed to explore freely for 10 min and the time spent exploring each object was recorded. Throughout the experiments, the objects were used in a counterbalanced manner in terms of their physical complexity and emotional neutrality. A preference index, a ratio of the amount of time spent exploring any one of the two objects (training session) or the novel object (retention session) over the total time spent exploring both objects, was used to measure cognitive function. To eliminate the influence of the last behavioral test, the objects were changed each time.
Spontaneous alternation in a Y-maze test
This behavioral test was performed at 6 and 13 months of age according to previous reports (20
, 21)
. The maze was made of black painted wood; each arm was 40 cm long, 12 cm high, 3 cm wide at the bottom, and 10 cm wide at the top. The arms converged at an equilateral triangular central area that was 4 cm at its longest axis. Each mouse was placed at the center of the apparatus and allowed to move freely through the maze during an 8 min session. The series of arm entries was recorded visually. Alternation was defined as successive entry into the three arms on overlapping triplet sets. Alternation behavior (%) was calculated as the ratio of actual alternations to possible alternations (defined as the number of arm entries minus two) multiplied by 100. To eliminate the influence of the last behavioral test, we used two Y-mazes in different rooms without changing the experimental conditions (size of apparatus, brightness of room, temperature, and humidity).
Morris water maze test
This behavioral test was performed at 6 and 13 months of age according to previous reports (22
, 23)
, with minor modifications. The Morris water maze test was conducted in a circular pool 1.2 m in diameter and filled with water at a temperature of 22 ± 1°C. A hidden platform (7 cm in diameter) was used. The mice were given two trials (one block) for 10 consecutive days, during which the platform was left in the same position. The time taken to locate the escape platform (escape latency) was determined in each trial by using the Etho Vision system (Neuroscience Idea Co. Ltd., Osaka, Japan). Three hours after the last training trial, the mice were given a transfer test without the platform and allowed 60 s to search the pool. To eliminate the influence of the last behavioral test, we used two water mazes in different rooms without changing the experimental conditions (size of apparatus, brightness of room, water temperature and depth). Mice that exhibited odd behavior such as spinning, lack of swimming, or staying close to the periphery in the Morris water maze test at the age of 6 months were excluded from all behavioral experiments.
Cued and contextual fear conditioning tests
Cued and contextual fear conditioning tests were performed at 13 months of age according to previous reports (19
, 24)
, with minor modifications. For measuring basal levels of freezing response (preconditioning phase), mice were individually placed in a neutral cage (17x27x12.5 high cm) for 1 min, then in the conditioning cage (25x31x11 high cm) for 2 min. For training (conditioning phase), mice were placed in the conditioning cage, then a 15 s tone (80 dB) was delivered as a conditioned stimulus. During the last 5 s of the tone stimulus, a foot shock of 0.6 mA was delivered as an unconditioned stimulus through a shock generator (Neuroscience Idea Co. Ltd.). This procedure was repeated four times with 15 s intervals. Cued and contextual tests were carried out 1 day after fear conditioning. For the cued test, the freezing response was measured in the neutral cage for 1 min in the presence of a continuous-tone stimulus identical to the conditioned stimulus. For the contextual test, mice were placed in the conditioning cage and the freezing response was measured for 2 min in the absence of the conditioned stimulus.
Biochemical analysis
Experimental design
Ten days after the behavioral test battery, the mice were sacrificed by CO2 asphyxiation, and their brains were removed and cut in half sagitally. The hemispheres were randomly chosen from each group for each biochemical analysis.
Immunohistochemistry
Free-floating sections were fixed for 15 min with 70% formic acid or 4% paraformaldehyde in 0.1M phosphate buffer and rinsed with PBS-Triton before being incubated in 0.3% H2O2 in methanol for 30 min. Sections were then incubated at room temperature for 2 h with the antibody indicated below, washed with PBS-Triton, incubated with a secondary goat anti-mouse or rabbit antibody for 2 h, washed with PBS-Triton again, and stained by the avidin-biotin HRP/DAB method. Primary antibodies against CD3e, CD4, CD8, CD19, and CD11b (BD Biosciences PharMingen, San Jose, CA, USA; 1:50), synaptophysin (Chemicon International, Temecula, CA, USA; 1 mg/ml), and Iba-1 for microglia (kindly provided by Dr. U. Imai, NCNP, Tokyo, Japan; 1 µg/ml) were used. Aß plaque-containing sections were stained with rabbit polyclonal anti pan Aß body (Biosource, Camarillo, CA, USA; 0.14 µg/ml).
A quantitative analysis of amyloid burden was performed as described previously (6)
in three different regions of the brain (AAV/control-treated Tg2576 mice, n=7; AAV/Aß-treated Tg2576 mice, n=6): the hippocampus, the frontal cortex, and the parietal association cortex. Images were projected from an Olympus Vanox microscope onto a computer screen through a 3CCD Fujix Digital Camera. Images were captured and analyzed with an image analysis system (Mac Scope, Mitani Co., Fukui, Japan). Aß burden was expressed as the percentage of brain tissue occupied by Aß deposits. Three immunolabeled sections were analyzed per mouse, and the average of the individual measurements was used to calculate group means.
Sample preparation for detection of insoluble and soluble Aß
The frozen hemispheres of Tg2576 brain tissue were obtained from each animal and homogenized with a homogenizer in TBS containing protease inhibitor cocktail (Complete; Roche Diagnostics, Mannheim, Germany) with 20 µg/ml pepstatin A (Roche Diagnostics), then centrifuged at 100,000 g for 1 h at 4°C using an Optima TLX ultracentrifuge (Beckman Coulter Inc., Fullerton, CA, USA). The pellets were homogenized in TBS containing 2% SDS and protease inhibitor cocktail (Complete; Roche Diagnostics), then centrifuged at 100,000 g for 1 h at 25°C after 15 min of incubation at 37°C. The supernatant and pellet correspond to the soluble and insoluble fraction, respectively.
ELISA for detection of insoluble Aß
Using highly specific antibodies and a sensitive sandwich enzyme-linked immunoabsorbent assay (ELISA), we quantified insoluble Aß40 and Aß42 in brain homogenate (AAV/control-treated Tg2576 mice, n=8; AAV/Aß-treated Tg2576 mice, n=8) as described previously (25)
. The pellet (referring to the sample preparation) was washed, then extracted further with 70% formic acid and centrifuged at 100,000 g for 1 h. The supernatants of insoluble 70% formic acid extracts were neutralized with 1M Tris-HCl, pH8.0 at a dilution of 1:20. For quantification of the insoluble fractions, we used a ß-amyloid ELISA kit (Wako, Osaka, Japan). The supernatant was diluted with standard dilution buffer at 1:2000 (Aß40) or 1:400 (Aß42) and measured according to the manufacturer’s instructions. The values obtained were corrected with the wet weight of each brain hemisphere sample and expressed as pmol/g brain.
Western blot analysis for detection of Aß oligomers in the soluble fraction
For analysis of Aß oligomers in the soluble fractions of mouse brain tissues (AAV/control-treated non-tg mice, n=4; AAV/control-treated Tg2576 mice, n=12; AAV/Aß-treated Tg2576 mice, n=10), the supernatants (referring to the sample preparation) were electrophoresed on 15/25% gradient SDS-PAGE gels (Daiichi Pure Chemicals, Tokyo, Japan) and transferred onto 0.2 µm nitrocellulose membranes at 200 mA for 1 h. Filters were blocked with 5% nonfat milk in a 20 mM Tris-HCl, pH7.4 containing 150 mM NaCl and 0.05% Tween 20 (TBS-T). After the membranes had been washed in TBS-T, the monoclonal anti-Aß antibody 6E10 (Senetek PLC, Napa, CA, USA) was used to probe the blots. Bound antibody was visualized using horseradish peroxidase (HRP) -conjugated anti-mouse IgG (at 1:10,000) and ECL+ detection (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). The intensity of bands on the film was analyzed by densitometry using the ATTO Densitograph Software Library Lane Analyzer (ATTO, Tokyo, Japan).
Berlin blue stain
To detect the hemorrhagic lesion in the mouse brain sections, we used Berlin blue stain. Briefly, brain sections were stained in potassium ferrocyanide solution (2% potassium ferrocyanide: 2% hydrochrolic acid=1 vol:1 vol; Muto Pure Chemicals, Tokyo, Japan) for 30 min. After washing in distilled water, sections were incubated in Kernechtrot stain solution (Muto Pure Chemicals, Tokyo, Japan) for 5 min.
Serum antibodies against Aß42
Anti-human Aß antibody titers in serum (naive Tg2576 mice, n=14; AAV/control-treated Tg2576 mice, n=14; AAV/Aß-treated Tg2576 mice, n=14) were quantified by a sandwich ELISA. Microtiter ELISA plates were coated overnight at 4°C with 4 µg/ml of synthetic human Aß1–42 in 55 mM NaHCO3, pH8.3. Plates were washed twice with washing buffer and blocked with 1% BSA and 2% normal goat serum in PBS for 2 h. Plates were washed twice and incubated with mouse serum samples diluted 1:500 in blocking buffer for 2 h while shaking. Plates were washed and incubated in HRP-conjugated goat anti-mouse IgG for 2 h. Plates were washed and analyzed colorimetrically after incubation with the chromogen substrate 3,3',5,5'-tetramethylbenzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). The absorbance of each well at 450 nm was read with the microplate reader. Serum IgG isotypes were determined using mouse immunoglobulin isotyping ELISA kit (BD Bioscience PharMingen, San Jose, CA, USA) according to the manufacturer’s instructions.
Statistic analysis
All results were expressed as the mean ± SE for each group. The difference between groups was analyzed with a 1-way, 2-way, or repeated ANOVA, followed by the Student-Newman-Keuls multiple range test. The Student’s t test was used to compare two sets of data. Pearson’s correlation coefficient test was used to examine the relationship between cognitive function and Aß*56 contents in the hippocampus.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Effect of oral vaccination on the accumulation of Aß in Tg2576 mice
We investigated the effect of an oral vaccination against Aß on the development of an AD-like neuropathology in Tg2576 mice treated at the age of 10 months. Quantitative image analyses were done in three different brain regions of AAV/Aß- or AAV/control-vaccinated Tg2576 mice at 13 months of age. Oral vaccination with AAV/Aß in Tg2576 mice resulted in a marked reduction in the deposition of Aß compared with the control group (Fig. 1
A). The mean value of Aß burden in the AAV/Aß-vaccinated group (frontal lobe: 0.06±0.065%, parietal lobe: 0.15±0.22%, hippocampus: 0.11±0.07%) was significantly reduced compared with that of the AAV/control-vaccinated group (frontal lobe: 1.68±0.84%, parietal lobe: 0.83±0.64%, hippocampus: 0.61±0.52%) (Fig. 1B
). The contents of insoluble Aß40 and Aß42 in the hippocampus of Tg2576 mice were quantified by using the sandwich ELISA. Oral vaccination with AAV/Aß significantly reduced the contents of insoluble Aß40 and Aß42 compared with those in the control group in Tg2576 mice (Fig. 1C
). The trimeric, tetrameric, hexameric, nonameric, and dodecameric (Aß*56) Aß oligomers in the soluble fraction of the hippocampus of Tg2576 mice were detected by using Western blot (Fig. 1D
). Oral vaccination with AAV/Aß in Tg2576 mice resulted in a marked reduction in the contents of Aß*56 and the nonameric Aß oligomer, but not other (trimeric, tetrameric, and hexameric) Aß oligomers (Fig. 1D
).
|
Effect of oral vaccination on the impaired learning and memory in Tg2576 mice
Object recognition in a novel object recognition test
We evaluated visual recognition memory at the age of 6, 10, and 13 months in a novel object recognition test. During the training session, there were no significant differences in exploratory preference between the two objects and total exploratory time among the groups (data not shown), suggesting that all groups of mice have the same levels of motivation, curiosity, and interest in exploring novel objects.
For the retention session, there was no difference in the level of exploratory preference for the novel objects between non-tg and Tg2576 mice at the age of 6 months (Fig. 2
A). However, the level of exploratory preference for the novel objects in Tg2576 mice at the age of 10 and 13 months was significantly decreased compared with that in the non-tg mice at the age of 10 and 13 months (Fig. 2B, C
), indicating an impairment of recognition.
|
When Tg2576 mice were vaccinated with AAV/Aß at the age of 10 months, at 13 months they spent a significantly longer time exploring the novel object than the AAV/control-vaccinated Tg2576 mice, indicating that AAV/Aß improved the impairment of recognition memory (Fig. 2C
). There were no effects of the vaccination on the preference for the novel object in non-tg mice (Fig. 2C
). The significant inverse relationship between cognitive ability and Aß*56 contents in the hippocampus was observed in the novel object recognition test (Fig. 2D
).
Spontaneous alternation in a Y-maze test
We evaluated short-term memory at the age of 6 and 13 months in a Y-maze test. There was no significant difference in the number of arm entries among the groups (data not shown), suggesting that all mice have the same levels of motivation, curiosity, and motor function. At the age of 6 months, Tg2576 mice showed significantly reduced spontaneous alternation behavior in a Y-maze test compared with non-tg mice (Fig. 3
A). At 13 months, the AAV/control-vaccinated Tg2576 mice also showed a significant reduction in spontaneous alternation behavior compared with AAV/control-vaccinated non-tg mice (Fig. 3B
). When Tg2576 mice were vaccinated with AAV/Aß at 10 months of age, their alternation behavior at 13 months was indistinguishable from that of AAV/control-vaccinated non-tg mice and significantly increased compared with that of AAV/control-vaccinated Tg2576 mice (Fig. 3B
). The alternation behavior of AAV/Aß-vaccinated non-tg mice was not different from that of AAV/control-vaccinated non-tg mice (Fig, 3B
). The significant inverse relationship between cognitive ability and Aß*56 contents in the hippocampus was observed in the Y-maze test (Fig. 3C
).
|
Reference memory in the Morris water maze test
We evaluated reference memory at the age of 6 and 13 months in a Morris water maze test. At the age 6 months, both non-tg and Tg2576 mice managed to learn the position of the hidden platform and there was no difference between these mice in reference memory (Fig. 4
A, B). When the transfer test was carried out after the 10th training trial, both mice again searched preferentially in the trained quadrant (Fig. 4C
).
|
At the age of 13 months, the AAV/control-vaccinated non-tg mice managed to learn the position of the hidden platform (Fig. 5
A, B). However, the AAV/control-vaccinated Tg2576 mice took a significantly longer time and distance to reach the platform than the AAV/control-vaccinated non-tg mice (Fig. 5A, B
), indicating an impairment of reference memory. When Tg2576 mice were vaccinated with AAV/Aß at the age of 10 months, at 13 months they took significantly shorter times and distances to reach the platform than the AAV/control-vaccinated Tg2576 mice (Fig. 5A, B
).
|
In the transfer test, the AAV/control- and AAV/Aß-vaccinated non-tg mice searched preferentially in the trained quadrant, but the AAV/control-vaccinated Tg2576 mice did not (Fig. 5C
), whereas the AAV/Aß-vaccinated Tg2576 mice preferentially searched in the trained quadrant (Fig. 5C
). The significant inverse relationship between cognitive ability and Aß*56 contents in the hippocampus was observed in the probe trial of Morris water maze test (Fig. 5D
). The decreased ability of the AAV/control-vaccinated Tg2576 mice did not reflect a loss of swimming ability or motivation, because swimming speed and distance in the transfer test were similar to values for other mice (data not shown).
Associative learning in the cued and contextual fear conditioning tests
Last, we evaluated associative learning at the age of 13 months in a conditioned fear learning test. In the preconditioning phase (training), the mice hardly showed any freezing response. There were no differences in basal levels of freezing response between the groups (data not shown). In the contextual learning test, the AAV/control- and AAV/Aß-vaccinated non-tg mice showed a marked contextual freezing response 24 h after fear conditioning (Fig. 6
A). There was no difference in freezing response between the two groups of non-tg mice. However, the AAV/control-vaccinated Tg2576 mice exhibited less of a freezing response in the contextual tests (Fig. 6A
), indicating an impairment of associative learning. The AAV/Aß-vaccinated Tg2576 mice were indistinguishable from the AAV/control-vaccinated non-tg mice, and treatment significantly reversed the contextual freezing response compared with AAV/control-vaccinated Tg2576 mice. In the cued learning test, there was no difference in the cued freezing response 24 h after fear conditioning among the groups (Fig. 6B
). The significant inverse relationship between cognitive ability and Aß*56 contents in the hippocampus was observed in the context-dependent but not cue-dependent test (Fig. 6C, D
). No alterations of nociceptive response were found in any of the mutant mice: there was no difference in the minimal current required to elicit flinching/running, jumping, or vocalization among the mice (data not shown).
|
Effect of oral vaccination on Aß-induced microglial attraction and synaptic degeneration
We investigated the effect of an oral vaccination against Aß on Aß-induced microglial attraction and synaptic degeneration in Tg2576 mice that were treated at the age of 10 months. In the frontal lobe of AAV/control-vaccinated Tg2576 mice at 13 months of age, staining for CD11b- and Iba-1, markers of microglia, showed there were many microglia surrounding Aß plaques (Fig. 7
A, B). Oral vaccination with AAV/Aß in Tg2576 mice decreased the number of microglia associated with Aß plaques in the frontal lobe at the age of 13 months (Fig. 7D, E
).
|
Synaptophysin (a marker of presynaptic vesicles) immunoreactivity was shrunken and aggregated within the Aß plaques in the frontal lobe of AAV/control-vaccinated Tg2576 mice at 13 months of age (Fig. 7C
). Synaptophysin-stained sections of the frontal lobe of AAV/Aß-vaccinated Tg2576 mice revealed that AAV/Aß decreased the number of plaques containing dystrophic neurites (Fig. 7F
). These ameliorating effects of the AAV/Aß vaccination on the Aß-induced microglial attraction and synaptic degeneration in Tg2576 mice were observed not only in the frontal lobe, but also in other regions of the brain (hippocampus and pariental lobe) (data not shown).
No lymphocytic infiltration and microhemorrhages in the brain of AAV/Aß-vaccinated Tg2576 mice
Lymphocytic infiltration is observed in the brain of vaccinated mice (26
, 27)
. We investigated whether lymphocyte infiltration occurred in the brain of Tg2576 mice treated with AAV/Aß at the age of 10 months. We evaluated changes in lymphocytic infiltration by staining with hematoxylin and eosin (H&E) (Fig. 8
A, B) and by changes in immunohistochemical staining with antibodies against CD3, CD4, CD8, and CD19, markers of mature T-lymphocytes, helper T-lymphocytes, cytotoxic T-lymphocytes, and B-lymphocytes, respectively (Fig. 8E-L
). There were no histological or immunohistochemical differences in the frontal lobes between AAV/control-vaccinated and AAV/Aß-vaccinated Tg2576 mice at the age of 13 months (Fig. 8A-L
).
|
The brain microhemorrhage associated with the increase in vascular amyloid levels (28)
is reported as an adverse side effect of passive immunotherapy in APP transgenic mice (29
, 30)
. However, we found neither microhemorrhage by H&E or Berlin blue staining (Fig. 8B, D
) nor an increase of vascular amyloid levels (data not shown) in the AAV/Aß-vaccinated Tg2576 mice.
Similar results were obtained for other regions of the brain (hippocampus and parietal lobes) (data not shown).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Tg2576 mice, an animal model of AD with overexpression of human APP695 with the Swedish mutation, show a subtle age-related formation of Aß plaques and progressive memory deficits (31)
. We confirmed the progressive impairment of object recognition memory in the object recognition test, short-term memory in the Y-maze test, reference memory in the water maze test, and associative learning in the contextual conditioned fear learning test in Tg2576 mice. It is unlikely that the impaired performance of Tg2576 mice in learning and memory tests is due to changes in motivation or sensorimotor function, since the motivation for each behavioral test is different, and different skills are required for a good performance in each test. There were no differences in locomotor activity, total time spent exploring objects in the novel object test, or swimming speed in the Morris water maze test between nontransgenic and Tg2576 mice.
Aß is distinguished biochemically by solubility properties in a detergent buffer (32)
. Insoluble Aß, which constructs senile plaques, is not detected until 6 months of age, but Aß plaques are observed at 9–12 months in the brain of Tg2576 mice (32)
. Insoluble Aß content correlates inversely with performance of the water maze task in Tg2576 mice (15)
. In the present study, Aß plaques were observed in the hippocampus and perirhinal cortex of Tg2576 mice at 13 months of age. Recognition memory in the novel object test (33
, 34)
, reference memory in the Morris water maze test (35)
, and associative learning in the contextual, but not cued conditioned fear learning test (36)
, are dependent on the hippocampus and/or perirhinal cortex. Taken together, our results suggest that the hippocampus- and/or perirhinal cortex-dependent behavioral tests are impaired by an accumulation of insoluble Aß, since in Tg2576 mice abnormal test results were obtained at 13 but not at 6 months of age, and cued fear conditioning dependent on the amygdala (36)
was not affected even at 13 months. When Tg2576 mice were treated with AAV/Aß at the age of 10 months, these cognitive impairments, the formation of Aß plaques, and the levels of insoluble Aß were attenuated at 13 months. We demonstrated that AAV/Aß alleviates these cognitive impairments via removal of insoluble Aß.
There is strong evidence that insoluble Aß is responsible for age-related memory decline. However, soluble Aß oligomers, but not fibrils or monomers, have been considered responsible for cognitive dysfunction prior to the formation of Aß plaques (37)
. In a recent study, cognitive dysfunction was found to be modest but significant in Tg2576 mice at 6 months of age, caused by the extracellular accumulation of Aß*56, a 56 kDa-soluble Aß assembly (16)
. We also found an impairment of short-term memory in the Y-maze test among Tg2576 mice at 6 and 13 months of age and an accumulation of hippocampal Aß*56 in the Tg2576 mice at 13 months. When Tg2576 mice were treated with AAV/Aß at 10 months of age, impairments of short-term memory and an accumulation of hippocampal Aß*56 at 13 months were attenuated. A significant inverse relationship between cognitive ability and Aß*56 contents in the hippocampus was observed in all the behavioral tests, including a Y-maze test at the age of 13 months. Thus, these findings suggested that the impairment of short-term memory in the Y-maze test is caused by Aß*56 and that AAV/Aß attenuates the impairment of short-term memory by eliminating the accumulated Aß*56.
Oral vaccination with AAV/Aß significantly reduced the contents of Aß*56 and nonameric Aß oligomer but not other Aß oligomers in Tg2576 mice. It is suggested that the vaccination of AAV/Aß inhibits assembly of soluble Aß oligomers since it binds Aß monomer and oligomers. Thus, Aß*56 showed the highest reduction among soluble Aß oligomers in the AAV/Aß-treated Tg2576 mice. Many reports of immunotherapy for AD have focused on the elimination of Aß plaques whereas Tg2576 mice exhibit very low levels of plaque deposition, apparently <1% for both the parietal cortex and hippocampus. It is hard to believe that reducing the number of plaques from 
1% to 0.1% would have such dramatic and long-lasting effects on abnormal behavior. Our finding has demonstrated that AAV/Aß could remove not only insoluble Aß but also soluble Aß oligomers.
Many histological studies have provided evidence that microglia surround Aß plaques in AD patients (38)
and APP transgenic mice (39)
. In the present study, microglial markers such as CD11b and Iba-1 showed that microglia are attracted to Aß plaques in Tg2576 mice at the age of 13 months. When Tg2576 mice were treated with AAV/Aß at 10 months of age, the microglial attraction disappeared accompanied by the clearance of Aß at 13 months. Microglial attraction is hypothesized to play a key role in the pathophysiology of AD, because Aß-activated microglia secrete proinflammatory products such as reactive oxygen species, cytokines, and neurotoxins (40
, 41)
. However, microglia infiltrate and restrict the formation of Aß plaques via phagocytosis in APP transgenic mice (39)
and a vaccination with Aß facilitates this phagocytosis through the production of antibody-Aß complexes (6)
. We previously observed an elevation in the level of anti-human Aß antibody in serum and activation of microglia in AAV/Aß-vaccinated Tg2576 mice 1 month after vaccination (14)
. Therefore, microglia in Tg2576 mice have little ability to remove Aß plaques through phagocytosis, but an AAV/Aß vaccination facilitates phagocytosis via binding of the antibody to Aß. We could not detect an increase in the number of activated microglia in AAV/Aß-vaccinated Tg2576 mice aged 13 months. Because the microglial activation induced by immunotherapy for Aß is transient (28)
, we could not detect it after the elimination of Aß deposits in AAV/Aß-vaccinated Tg2576 mice.
Degeneration of synapses along with the associated presynaptic protein synaptophysin is another cardinal histological feature of the brains of AD patients, and strongly correlates with cognitive decline (42)
. In the present study, we observed that synaptophysin immunoreactivity was shrunken and aggregated within the Aß plaques in Tg2576 mice at the age of 13 months. When Tg2576 mice were treated with AAV/Aß at 10 months of age, presynaptic degeneration was not observed. Axonally transported APP increases the amount of Aß that is released from presynaptic sites and deposited in extracellular plaques (43)
. APP and synaptophysin were reported to colocalize at the growth cones of developing neurons in culture (44)
. These reports have indicated that deposits of Aß play an important role in the degeneration of presynaptic structures, whereas vaccination with AAV/Aß ameliorates this presynaptic degeneration through removal of Aß.
We used a recombinant AAV for immunotherapy against Aß. This recombinant has several features that make it useful for immunotherapy. 1) AAV is resistant to extremes of temperature and pH and to solvents, making it particularly suitable as an orally delivered vector (45)
. We previously confirmed that orally administered AAV infects epithelial cells of the upper gastrointestinal tract without spreading to other organs (e.g., heart, lung, spleen) (14)
. 2) AAV can trigger enough long-term gene expression for immunity against Aß to be acquired. Transduction of AAV is observed in intestinal cells at >21 wk after treatment (14)
. In AAV/Aß-treated Tg2576 mice, IgG antibodies against Aß were detected in serum at 1 month, and remained at elevated levels for >6 months after vaccination (14)
. Thus, single administration of AAV/Aß can alleviate behavioral and biochemical abnormalities in Tg2576 mice. 3) AAV is safe, since wild AAV is not pathogenic to humans or other species (46)
and adeno-associated viral DNA normally does not integrate into the cellular genome; rather, it remains in the episome. On the other hand, the serious side effects observed in a phase II clinical trial of AN-1792 might have been induced by the Th-1, cytotoxic T-lymphocyte response (11)
, which may have been elicited by both the full-length Aß and the QS-21 adjuvant that promotes a cell-mediated Th-1-type immune response (47)
. AAV/Aß can generate antibodies and remove Aß plaques without lymphocytic infiltration (14)
, since it does not contain an adjuvant and the gut immune system suppresses Th-1-type responses and enhances Th-2-type responses (48)
. In fact, the anti-human Aß antibody in the serum of AAV/Aß-treated Tg2576 mice was significantly increased vs. that of AAV/control-treated Tg2576 mice at the age of 13 months; isotypes of the antibodies were identified by ELISA as IgG1
or IgG2b, suggesting a Th-2 response (Supplemental Data 1). No proliferation of T-lymphocytes in response to Aß was detected in splenocytes isolated from AAV/Aß-vaccinated Tg2576 mice (14)
, and we did not detect any differences between AAV/control- and AAV/Aß-vaccinated Tg2576 mice in the histological staining with H&E or immunohistochemical staining with antibodies against CD3, CD4, CD8, and CD19.
Amyloid angiopathy-associated microhemorrhage is observed in PDAPP mice with passive immunotherapy over the age of 22 months (30)
. Cotreatment with pertussis toxin (26)
and crossbreeding with interferon-
transgenic mice (27)
enhance the incidence of active immunotherapy-induced encephalitis. Although we need to conduct more detailed experiments using these models to ascertain that this would be predictive of what would happen in humans, the results suggest that oral administration of AAV/Aß may be safe for treating AD.
In conclusion, the development of a safe and effective immunotherapy for AD will require providing specific and adequate anti-Aß antibody responses sufficient for therapeutic benefit while eliminating adverse T-lymphocyte-mediated autoimmune responses. We demonstrated that oral administration of AAV/Aß reduced the accumulation of Aß and attenuated cognitive and histological abnormalities in Tg2576 mice without lymphocytic infiltration or microhemorrhage in the brain. Our results suggest that AAV/Aß may be a safe and effective immunotherapy for AD.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication November 17, 2006. Accepted for publication February 1, 2007.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
