|
|
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Article |
Deletion of tumor necrosis factor death receptor inhibits amyloid ß generation and prevents learning and memory deficits in Alzheimer's mice
Correspondence to Yong Shen: yong.shen{at}sunhealth.org
The tumor necrosis factor type 1 death receptor (TNFR1) contributes to apoptosis. TNFR1, a subgroup of the TNFR superfamily, contains a cytoplasmic death domain. We recently demonstrated that the TNFR1 cascade is required for amyloid ß protein (Aß)–induced neuronal death. However, the function of TNFR1 in Aß plaque pathology and amyloid precursor protein (APP) processing in Alzheimer's disease (AD) remains unclear. We report that the deletion of the TNFR1 gene in APP23 transgenic mice (APP23/TNFR1–/–) inhibits Aß generation and diminishes Aß plaque formation in the brain. Genetic deletion of TNFR1 leads to reduced ß-secretase 1 (BACE1) levels and activity. TNFR1 regulates BACE1 promoter activity via the nuclear factor-
B pathway, and the deletion of TNFR1 in APP23 transgenic mice prevents learning and memory deficits. These findings suggest that TNFR1 not only contributes to neurodegeneration but also that it is involved in APP processing and Aß plaque formation. Thus, TNFR1 is a novel therapeutic target for AD.
Abbreviations used in this paper: Aß, amyloid ß protein; AD, Alzheimer's disease; ANOVA, analysis of variance; APP, amyloid precursor protein; BACE1, ß-secretase 1; CAA, cerebral amyloid angiopathy; IDE, insulin degradation enzyme; NEP, neprilysin; NF-
B, nuclear factor B; TNFR1, TNF type 1 death receptor; vWF, von Willebrand factor.
 |
Introduction |
|---|
 TopAbstract Introduction ResultsDiscussionMaterials and methodsReferences |
|---|
4.5 million Americans, and this number will continue to grow. By 2050, the number of individuals with AD could range from 11.3 to 16 million (Hebert et al., 2003). The pathogenesis underlying AD remains unclear, and it is controversial whether AD results from a primary abnormality in amyloid precursor protein (APP) or deregulation of the inflammatory system (Weiner and Selkoe, 2002), although these two possibilities are not mutually exclusive. Several lines of evidence implicate abnormal processing of APP, which is cleaved by two enzymes, ß-secretase 1 (BACE1) and 
-secretase, to generate excessive amyloid ß protein (Aß), as a potential cause of AD (Selkoe, 2003; Tanzi and Bertram, 2005). In the past decade, transgenic mice have been generated that overexpress mutant APP and display Aß-related lesions (Hsiao et al., 1995). Many of these mouse models exhibit amyloid plaque-predominant aspects of AD (Terry et al., 1987; Tiraboschi et al., 2004), including Aß plaque formation, cerebral amyloid angiopathy (CAA), and inflammation, but not 
pathology.
The TNF death receptor belongs to the TNFR superfamily, which includes >20 cell surface receptors. When the TNF type 1 death receptor (TNFR1) binds to its ligand, TNF
, the ligand–receptor complex triggers apoptotic pathways by recruiting a TNFR-associated death domain protein and/or a Fas-associated death domain protein/mediator of receptor-induced toxicity, two intracellular adaptor proteins (Boldin et al., 1995). The receptor-induced multimerization of a Fas-associated death domain protein leads to caspase activation, which causes degradation of specific target proteins, ultimately damaging cell integrity (Ashkenazi and Dixit, 1998).
To find out whether TNFR1 could have effect on Aß production as well as APP processing, we specifically chose transgenic APP23 mice in our experiments, which express a mutant APP that results in extensive Aß plaque formation. Here we show that fewer Aß plaques and Aß-related lesions develop in Alzheimer's transgenic mice with genetic deletion of TNFR1. Detailed analyses showed decreased Aß generation, less neuronal loss, and alleviated Aß-related memory deficits. Our data indicates that TNFR1 might be a potential and novel therapeutic target for AD.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
|
The size of Aß plaques also indicates the severity of Aß pathology (Zhou et al., 2005). We used morphometric analyses on the brain sections immunostained with Aß antibody 6E10. Results showed that both large (>20 µm diameter)- and medium (10–20 µm diameter)-sized Aß plaques in the entorhinal cortex (Fig. 1 F) and hippocampus (Fig. 1 G) were significantly reduced in APP23/TNFR1–/– mice at 12 and 24 mo of age, indicating that in APP23/TNFR1–/– mice, Aß pathology was alleviated not only by reducing the overall Aß plaque number, but also by decreasing plaque size.
TNFR1 deletion reduces CAA
CAA has been reported to have both positive and negative correlations with AD pathology (Cohen et al., 1997; Thal et al., 2003; Tian et al., 2003). It has been shown that CAA in APP23 transgenic mice is strikingly similar to that of human CAA (Calhoun et al., 1999). To find out whether genetic deletion of TNFR1 can relieve CAA in APP23 mice, the deposition of Aß in the vascular wall was examined by double immunostaining with antibodies against ß-smooth muscle actin (a vascular smooth muscle marker; Skalli et al., 1986) or von Willebrand factor (vWF; an endothelial cell marker; Shyu et al., 2006) and anti-Aß40 antibody. We found that at 24 mo of age, APP23 mice display CAA predominant in cortical, hippocampal, and thalamic vessels; Aß40 formed a continuous ring-like shape within the vessel wall (Fig. 2, A and B), consistent with Calhoun et al. (1999).
However, there were very few Aß40 deposits within the vessels of APP23/TNFR1–/– (Fig. 2, C and D). Deposition of Aß on the vascular wall could not only increase the vulnerability of cerebral vessels but also increase the possibility of intracerebral hemorrhage (Vinters, 1987; Itoh et al., 1993; Winkler et al., 2001; Atwood et al., 2003). Our results showed little CAA progression in the brains of APP23/TNFR1–/– mice, suggesting that deletion of TNFR1 could reduce the risk of CAA.
|
|
|
TNFR1 deletion alters BACE1 activity and level
To examine whether the reduced amyloidosis in APP23/TNFR1–/– mice was caused by a reduction in abnormal APP metabolism, we examined the activity and expression levels of one key enzyme in APP processing, BACE1.
We first used an MCA-labeled BACE1 substrate (Yang et al., 2003; Li et al., 2004a) to examine BACE1 activity and found that BACE1 activity was significantly decreased in APP23/TNFR1–/– mice (Fig. 5 A). To find out whether the decreased BACE1 activity was due to a decrease in BACE1 levels, we measured BACE1 levels by sandwich ELISA (Yang et al., 2003) and Western blot (n = 3 for each group). We found that BACE1 levels in APP23/TNFR1–/– mice were indeed reduced in both Western blot and ELISA results (Fig. 5, B and C), indicating that reduced BACE1 activity in APP23/TNFR1–/– mice was caused by a reduction in the protein level. To further investigate whether reduced BACE1 protein level is caused by reduced BACE1 mRNA transcription, we performed RT-PCR to measure BACE1 mRNA levels and found that BACE1 mRNA was also decreased in APP23/TNFR1–/– mice (Fig. 5 D), indicating that the genetic deletion of TNFR1 reduced BACE1 mRNA levels and caused BACE1 activity to be down-regulated in APP23/TNFR1–/– mice.
|
regulates BACE1 transcription through the TNFR1–nuclear factor B (NF-B) pathway
To clarify how deletion of TNFR1 affects BACE1, we transfected 293 cells with a pB1P-A vector containing a BACE1 promoter (–1941 to +292) that was upstream of a luciferase reporter gene (Christensen et al., 2004; Sambamurti et al., 2004), and then treated these cells with different concentrations of TNF. We found that BACE1 promoter activity increased in a concentration-dependent manner (Fig. 6 A).
Blocking the interaction of TNF with the extracellular domain fragment of TNFR1 inhibited such elevation in BACE1 promoter activity (Fig. 6 A), indicating that TNF activates BACE1 promoter through TNFR1.
|
B is one of the major mediators of TNF-activated TNFR1 signaling (Hsu et al., 1995; Yang et al., 2002). A recent finding that multiple NF-B binding sites are located in the vicinity of BACE1 promoter (Sambamurti et al., 2004) suggests that NF-B may play an important role in regulating BACE1 transcription. To determine whether TNFR1 activates the BACE1 promoter through this pathway, we used the potent NF-B activation inhibitor 6-amino-4(4-phenoxyphenylethylamino) quinazoline (Tobe et al., 2003) to block NF-B signaling in TNF- treated 293 cells transfected with a BACE1 promoter luciferase reporter vector. Treating a pB1P-A transfected cell with an NF-B inhibitor significantly reduced TNF-induced BACE1 promoter activity (Fig. 6 B). A high concentration of NF-B inhibitor inhibited not only TNF-induced BACE1 promoter activity but also basal promoter activity (Fig. 6 B), indicating that NF-B may play a central role in regulating BACE1 transcription. Thus, the TNF-mediated activation of NF-B through TNFR1 represents a key part of this regulatory pathway.
These findings indicate that one mechanism underlying the regulation of BACE1 transcription may be through TNFR1-mediated activation of NF-B. We found significantly lower Aß as well as BACE1 levels in APP23/TNFR1–/– in older specimens (12 and 24 mo). One possible explanation is that when Aß deposits are lower (at both time points), there is less Aß to extract, therefore lower Aß levels might not be caused by the reduced BACE1 level. To examine whether the reduced Aß level is caused by the reduced BACE1 level, we measured Aß and BACE1 levels in APP23/TNFR1–/– mice at 6 mo of age, before Aß pathology can be observed. If TNFR1 affects the BACE1 level, it should also reduce the BACE1 level at this age. We first found that total Aß in APP23/TNFR1–/– mice was much lower than in APP23 mice (Fig. 7 A).
Both Aß40 and Aß42 levels in APP23/TNFR1–/– mice were also reduced. A Western blot showed a reduction of the BACE1 protein level in APP23/TNFR1–/– mice (Fig. 7 B). BACE1 RT-PCR showed a similar result to that of 12-mo-old mice; the BACE1 mRNA level was significantly lower in APP23/TNFR1–/– mice than in APP23 mice (Fig. 7 C). Together, these findings indicate that TNFR1 indeed regulates the BACE1 mRNA level, and that Aß reduction in APP23 mice after genetic deletion of TNFR1 is caused by decreased BACE1 levels.
|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
is one of the up-regulated inflammation factors in APP transgenic mice (Patel et al., 2005). Here we found that TNFR1 could directly regulate BACE1 transcription through NF-B, which is one of the major mediators of TNF-activated TNFR1 signaling (Hsu et al., 1995; Yang et al., 2002). Recent findings showed multiple NF-B binding sites located in the vicinity of BACE1 promoter (Sambamurti et al., 2004), suggesting that NF-B may play an important role in regulating BACE1 transcription. This is confirmed by our results that the NF-B inhibitor inhibits BACE1 promoter activity.
At 24 mo of age, we found significant neuronal loss in APP23 mice. However, Irizarry et al. (1997) did not observe neuronal loss in 16-mo-old APP transgenic mice (Tg2576) expressing the APPK670N/M671L mutation, the same mutation harbored by APP23 mice. This might be because the APP transgene is controlled by different promoters in APP23 and Tg2576 mice (Irizarry et al., 1997; Sturchler-Pierrat et al., 1997). TNFR1 deficiency ameliorates neuron loss in APP23 mice, consistent with our previous findings that TNFR1 overexpression increases the vulnerability of cultured hippocampal neurons to Aß-induced death and promotes neuronal degeneration (Li et al., 2004b). We also noticed that TNF plays different roles in neuronal death and survival via its distinct receptors, TNFR1 and TNFR2. Neuron loss in APP23 mice caused by "endogenous" Aß may be conducted through different signal transduction pathways. Moreover, Barger et al. (1995) showed that TNF can protect neurons derived from fetal brains against Aß toxicity. Our unpublished data show that TNFR1 is expressed at a low level, whereas TNFR2 is expressed at a high level in fetal neurons. This may explain why TNF is trophic in fetal neurons. Interestingly, Bruce et al. (1996) discovered that neurons from mice with a deficiency of both TNFR1 and TNFR2 are more sensitive to excitotoxic injury. This result is interesting because the finding suggests that there is a balance between TNFR1 and TNFR2 expression levels in neurons, and TNFR2 seems to be more critical and more sensitive to neurons.
Our behavioral analyses revealed that inactivation of TNFR1 rescued hippocampal-dependent learning and memory deficits displayed by young APP23 mice (Van Dam et al., 2003). A previous study reported that disruption of the BACE1 gene or PS1 in APP transgenic mice rescues memory deficits measured by social recognition and spatial alternation tasks (Saura et al., 2005). This is consistent with our findings in APP23/TNFR1–/– mice, presumably because TNFR1 depletion decreases Aß production and deposition, thereby reducing Aß-related memory deficits. The relatively normal performance of hippocampal-dependent memory tasks by APP23/TNFR1–/– mice is age related. At 6 mo of age, APP23/TNFR1–/– mice already performed hippocampal-dependent memory tasks better than APP23 mice. Furthermore, TNFR1 knockout mice exhibited normal synaptic transmission and plasticity in the Schaffer collateral pathway (unpublished data). Our results allow us to determine whether treating APP23 mice with anti-TNFR1 antibody or inhibitors of the TNFR1 signal transduction pathway could reduce BACE1 and cerebral Aß.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
APP23 and TNFR1–/– mice were crossed and their progeny were genotyped. An APP23/TNFR1+/– mouse was backcrossed with TNFR1–/– mice to produce APP23/TNFR1–/– mice. To maintain the heterozygous APP transgene in our mice, we crossed APP23 mice with wild-type C57BL/6 mice. For APP23/TNFR1–/– mice, we crossed APP23/TNFR1–/– with TNFR1–/– mice for three to five generations. Therefore, both APP23 and APP23/TNFR1–/– mice were APP23+/–. We used APP23/TNFR1–/– mice of the F3–F5 generation in our experiments.
Mice homozygous for the TNFR1 targeted mutation (formerly TNFR1, p55 deficient) show defects in resistance to intracellular pathogens and are resistant to the lethal effects of lipopolysaccharide administration in conjunction with D-galactosamine. Pulmonary inflammatory responses are diminished in p55-deficient mice. There are also defects in splenic architecture, formation of germinal centers, and liver regeneration. TNFR1- deficient mice display increased susceptibility to atherosclerosis when maintained on a high-fat diet (Peschon et al., 1998). No observations regarding any syndromes of the central nervous system have been made.
ELISA
APP23, APP23/TNFR1–/–, and wild-type mice (n = 10 per group) were killed at 12 and 24 mo of age, and one hemisphere of the brain was homogenized in homogenization buffer (250 mM sucrose, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 1 mM EGTA). An aliquot of the homogenate was dissolved in formic acid and neutralized with a neutralization buffer (1 mM Tris and 0.5 M Na2HPO4). Protein concentration was measured by protein assay (Bio-Rad Laboratories). For total Aß ELISA, the capture antibody was monoclonal anti-Aß antibody 4G8 (Chemicon), and the detection antibody was biotinylated monoclonal antibody anti-Aß 6E10 (AbD Serotec). Aß40 and Aß42 were measured with an Aß40 and Aß42 ELISA kit (Biosource International). The ELISA system has been extensively tested and no cross-reactivity between Aß40 and Aß42 was observed. Data are presented as means ± SD of four experiments.
BACE1 protein levels were measured by ELISA as described previously (Yang et al., 2003). The capture antibody was anti-BACE1 polyclonal antibody P1 (Yang et al., 2003) and the detection antibody was biotinylated anti-BACE1 polyclonal antibody P2 (Yang et al., 2003). TMB substrate was used to visualize the reaction product, which was read at OD450 with a microplate reader (Sigma-Aldrich). BACE1 protein (Amgen) was used as a standard. Data are presented as means ± SD of four experiments.
Western blot
Aliquots of brain homogenates from APP23, APP23/TNFR1–/–, and wild-type mice were further lysed with 1x RIPA buffer, and 50–150 µg of total protein was subjected to SDS-PAGE (8–12% acrylamide). Separated proteins were then transferred onto polyvinylidene fluoride membranes. The blots were probed with the following antibodies: anti-BACE1 monoclonal antibody (R&D Systems), anti-Aß (1–17) monoclonal antibody (clone 6E10, 1:2,000; Chemicon), anti-IDE polyclonal antibody (Oncogene Research Products), anti-NEP polyclonal antibody (Chemicon), and anti– ß actin antibody (Sigma-Aldrich).
Western blotting for Aß was performed as described previously (Wiltfang et al., 1997). To detect minute levels of Aß, formic acid–dissolved brain tissue was immunoprecipitated with anti-Aß polyclonal antibody (Zymed Laboratories) and subjected to SDS-PAGE using 10% acrylamide gels containing 8 M urea. Separated proteins were transferred onto polyvinylidene fluoride membranes. Aß40 and Aß42 were detected with monoclonal anti-Aß antibody 6E10. Synthetic Aß40 and Aß42 (Biosource International) were used as standards.
BACE1, IDE, and NEP activity
An aliquot of brain homogenates from APP23, APP23/TNFR1–/–, and wild-type mice was further lysed with a lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10% glycerol, and 0.5% Triton X-100). BACE1 enzymatic activity assays were performed by using synthetic peptide substrates containing BACE1 cleavage site (MCA-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-[Lys-DNP]-OH; Biosource International). BACE substrate was dissolved in DMSO and mixed with a 50-mM Hac and 100-mM NaCl, pH 4.1, reaction buffer. An equal amount of protein was mixed with 100 µl of substrate, and fluorescence intensity was measured with a microplate reader (BioTek) at an excitation wavelength of 320 nm and an emission wavelength of 390 nm.
IDE enzyme activity was measured as described previously (Song et al., 2003). In brief, brains were homogenized in 50 mM potassium phosphate buffer, pH 7.3, containing 200 µm PMSF and a proteinase inhibitor mix (Sigma-Aldrich). Samples were centrifuged and the supernatant fraction was used for IDE activity measurement. The hydrolysis of fluorogenic substrate peptides (2 µm Abz-GGFLRKHGQED-Dnp as substrate in 20 mM potassium phosphate buffer, pH 7.3) was measured by following an increase in fluorescence (excitation at 318 nm and emission at 419 nm) that occurred upon peptide bond cleavage. The max velocity of IDE activity was calculated by the first 20 min and indicated as fluorescence unit/min microgram protein.
For the in vitro NEP activity assay, mouse brains were homogenized in 100 mM MES buffer (pH 6.5) with proteinase inhibitors (Sigma-Aldrich). Homogenate was centrifuged at 20,000 g for 45 min to separate the membrane fraction and the supernatant was removed. The membrane pellet was resuspended in MES buffer and directly used in NEP activity assay as previously described (Li and Hersh, 1995).
RT-PCR
To compare BACE1 expression levels, we used the following primers for RT-PCR: mouse BACE1 forward primer, 5'-AGACGCTACACATCCTGGTG-3', and backward primer, 5'-CCTGGGTGTAGGGCACATAC-3'. The amplified BACE1 fragment was 146 bp. Mouse s18 was used as a loading control: forward primer, 5'-CAGAAGGACGTGAAGGATGG-3', and backward primer, 5'-CAGTGGTCTTGGTGTGCTGA-3'. The amplified mouse s18 fragment was 159 bp. Total RNA was extracted from the brains of 12-mo-old APP23 and APP23/TNFR1–/– mice (n = 5) using an RNA mini column kit (Invitrogen). RT-PCR was performed using a One- Step RT-PCR kit (Invitrogen) and the following PCR cycles: 50°C for 30 min, 94°C for 2 min, followed by 25 cycles at 94°C for 15 s, 49°C for 30 s, and 68°C for 1 min.
Cell transfection and luciferase assay
We transfected 293 cells with pB1P-A vector containing a BACE1 promoter (–1941 to +292) upstream from a luciferase reporter gene (Christensen et al., 2004) using lipofectamine (Invitrogen). After transfection, cells were treated with different concentrations of TNF (R&D Systems), extracellular domain of TNFR1 (R&D Systems), or NF-B inhibitor 6-amino-4(4-phenoxyphenylethylamino) quinazoline (Calbiochem; Tobe et al., 2003). Cells were collected 12 h after treatment, and a luciferase assay (Promega) was performed, according to the manufacturer's instructions. Luminescence intensity was measured with a microplate reader, normalized according to protein amount, and plotted as relative luminescence units per milligram of protein.
Immunohistochemistry and immunofluorescence
Immunohistochemistry was performed as previously described (Matsuoka et al., 2001). In brief, paraformaldehyde-fixed brains were quickly frozen, and then sectioned at 30 µm. Sections were incubated with either anti-Aß (6E10 clone or 4G8 clone, 1:1,000; Chemicon), anti-NeuN (MAB377, 1:400; Chemicon), anti-CD11b (MCA711, 1:500; AbD Serotec) and CD45 (MCA1388, 1:500; AbD Serotec), anti--smooth muscle actin (-SM actin, A2547, 1:400; Sigma-Aldrich), or anti-vWF (AB7536, 1:200; Chemicon). Secondary antibodies were applied with horse anti–mouse (for 6E10, NeuN detection, 1:1,000) and goat anti–rat (for CD45 or CD11b, 1:1,000) followed by a DAB substrate (Vector Laboratories). For immunofluorescence, fluorescent-labeling 488 (green) or 594 (red) secondary antibodies against rabbit IgG or mouse IgG were used (1:1,000; Invitrogen). A microscope (DMLS; Leica) with a 10x N PLAN and 20x and 40x PL FLUOTAR was used. Digital images were captured and processed by digital camera (Optronics) and MagnaFire software (version 2.1C; Optronics).
Quantitation of immunoreactive structures
30-µm serial sagittal sections through the entire rostrocaudal extent of the hippocampus were cut on a cryostat. Every 10th section was immunostained with anti-NeuN antibody. On all sections containing the hippocampus, we delineated the pyramidal cell layer CA1. The total number of neurons were obtained using unbiased stereology (Casas et al., 2004; Schmitz et al., 2004) and a microscope equipped with a digital camera (DEI-470; Optronics). For each section, we delineated a 400-µm2 area in CA1 and in the entorhinal cortex and counted all NeuN-immunoreactive cells within that 400-µm2 box. The mean sum of neurons was counted per animal (n = 10). We used the same method to count Aß-immunoreactive plaques (stained with 6E10) in the hippocampus and entorhinal cortex in a double blind test. We also measured the diameter of each counted plaque. Differences between groups were tested with Image-pro Plus Analysis (Media Cybernetics).
Hole-board memory task
As previously reported (Dodart et al., 2002), this task measured a mouse's ability to remember which one out of four equidistant holes was baited with food. Two photobeam apparatuses were used with a hole board for assessing directed exploration in mice for behavioral tests. A tested mouse (n = 10 for each group) was placed in the center of the hole-board and the number of nose pokes was automatically registered for 5 min. After 20 min, each animal was placed in a corner of the hole board and allowed to freely explore the apparatus for 5 min. The number of head dips, time spent head-dipping, and the number of rearings were recorded. A comprehensive cognitive performance was determined by calculating the mean number of correct pokes per trial that mouse made each day. Cognition was expressed as the percentage of correct pokes. The measurements in the hole-board test were analyzed by unpaired t test. In all cases the significance level was considered to be P < 0.05, and the very significant level was considered to be P < 0.01.
Object recognition task
The day before training, an individual mouse (n = 10 for each group) was placed into a training apparatus (a box the same size as described for the hole-board test) and allowed to habituate to the environment for 15 min. Training was initiated 24 h after habituation. A mouse was placed back into the training box containing two identical objects A and B (die or marble) and allowed to explore these objects. Among experiments, training times varied from 3.5 to 20 min. For each experiment, the same set of animals was used repeatedly with different sets of objects for each repetition. Five repetitions were performed on each set of mice. Each mouse was trained and tested no more than once per week, with a 1-wk interval between testing. Moreover, each experimental condition was replicated independently four times. In each experiment, the experimenter was blinded to the subjects during training and testing. To test memory retention, mice were observed for 10 min, 6 h, and 24 h after training. Mice were presented with two objects, one that was used during training, and thus was "familiar," and one that was novel. The test objects were divided into 10 sets of "training" plus "testing" objects, and a new set of objects was used for each training session. A recognition index was calculated for each mouse, expressed as the ratio (100TB)x(TA + TB), where TA and TB are the time spent during the second trial on subject A and subject B, respectively. To ensure that the discrimination targets did not differ in odor, the apparatus and the objects were thoroughly cleaned with 90% ethanol, dried, and ventilated for a few minutes after each experiment.
Statistical analyses
In general, analysis of variance models (ANOVA) were used to analyze behavioral data. Typically, the statistical models included two between-subjects variables, the genotype of mice (APP23 vs. APP23/TNFR1–/–) and age, and one within-subjects variable, such as blocks of trials. When ANOVAs with repeated measures were conducted, the Huynh-Feldt adjustment of levels was used for all within-subjects effects containing more than two levels to protect against violations of the sphericity/compound symmetry assumptions underlying this ANOVA model.
|
Acknowledgments |
|---|
Submitted: 8 May 2007
Accepted: 25 July 2007
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Ashkenazi, A., and V.M. Dixit. 1998. Death receptors: signaling and modulation. Science. 281:1305–1308.
Atwood, C.S., G. Perry, and M.A. Smith. 2003. Cerebral hemorrhage and amyloid-beta. Science. 299:1014.[Medline]
Barger, S.W., D. Horster, K. Furukawa, Y. Goodman, J. Krieglstein, and M.P. Mattson. 1995. Tumor necrosis factors alpha and beta protect neurons against amyloid beta-peptide toxicity: evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation. Proc. Natl. Acad. Sci. USA. 92:9328–9332.
Boldin, M.P., I.L. Mett, E.E. Varfolomeev, I. Chumakov, Y. Shemer-Avni, J.H. Camonis, and D. Wallach. 1995. Self-association of the "death domains" of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF and Fas/APO1 effects. J. Biol. Chem. 270:387–391.
Bourtchouladze, R., R. Lidge, R. Catapano, J. Stanley, S. Gossweiler, D. Romashko, R. Scott, and T. Tully. 2003. A mouse model of Rubinstein-Taybi syndrome: defective long-term memory is ameliorated by inhibitors of phosphodiesterase 4. Proc. Natl. Acad. Sci. USA. 100:10518–10522.
Brosnan-Watters, G., and D.F. Wozniak. 1997. A rotating holeboard procedure for testing drug effects on spatial learning and memory in mice. Brain Res. Brain Res. Protoc. 1:331–338.[CrossRef][Medline]
Bruce, A.J., W. Boling, M.S. Kindy, J. Peschon, P.J. Kraemer, M.K. Carpenter, F.W. Holtsberg, and M.P. Mattson. 1996. Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nat. Med. 2:788–794.[CrossRef][Medline]
Cagnin, A., D.J. Brooks, A.M. Kennedy, R.N. Gunn, R. Myers, F.E. Turkheimer, T. Jones, and R.B. Banati. 2001. In-vivo measurement of activated microglia in dementia. Lancet. 358:461–467.[CrossRef][Medline]
Calhoun, M.E., K.H. Wiederhold, D. Abramowski, A.L. Phinney, A. Probst, C. Sturchler-Pierrat, M. Staufenbiel, B. Sommer, and M. Jucker. 1998. Neuron loss in APP transgenic mice. Nature. 395:755–756.[CrossRef][Medline]
Calhoun, M.E., P. Burgermeister, A.L. Phinney, M. Stalder, M. Tolnay, K.H. Wiederhold, D. Abramowski, C. Sturchler-Pierrat, B. Sommer, M. Staufenbiel, and M. Jucker. 1999. Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid. Proc. Natl. Acad. Sci. USA. 96:14088–14093.
Casas, C., N. Sergeant, J.M. Itier, V. Blanchard, O. Wirths, N. van der Kolk, V. Vingtdeux, E. van de Steeg, G. Ret, T. Canton, et al. 2004. Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42 accumulation in a novel Alzheimer transgenic model. Am. J. Pathol. 165:1289–1300.
Christensen, M.A., W. Zhou, H. Qing, A. Lehman, S. Philipsen, and W. Song. 2004. Transcriptional regulation of BACE1, the beta-amyloid precursor protein beta-secretase, by Sp1. Mol. Cell. Biol. 24:865–874.
Cohen, D.L., P. Hedera, D.R. Premkumar, R.P. Friedland, and R.N. Kalaria. 1997. Amyloid-beta protein angiopathies masquerading as Alzheimer's disease? Ann. NY Acad. Sci. 826:390–395.[Medline]
Dodart, J.C., K.R. Bales, K.S. Gannon, S.J. Greene, R.B. DeMattos, C. Mathis, C.A. DeLong, S. Wu, X. Wu, D.M. Holtzman, and S.M. Paul. 2002. Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model. Nat. Neurosci. 5:452–457.[Medline]
Farris, W., S. Mansourian, Y. Chang, L. Lindsley, E.A. Eckman, M.P. Frosch, C.B. Eckman, R.E. Tanzi, D.J. Selkoe, and S. Guenette. 2003. Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc. Natl. Acad. Sci. USA. 100:4162–4167.
Frid, P., S.V. Anisimov, and N. Popovic. 2007. Congo red and protein aggregation in neurodegenerative diseases. Brain Res. Rev. 53:135–160.[CrossRef][Medline]
Galimberti, D., N. Schoonenboom, P. Scheltens, C. Fenoglio, F. Bouwman, E. Venturelli, I. Guidi, M.A. Blankenstein, N. Bresolin, and E. Scarpini. 2006. Intrathecal chemokine synthesis in mild cognitive impairment and Alzheimer disease. Arch. Neurol. 63:538–543.
Garcia, J. 1987. Sharing research results with patients: the views of care-givers involved in a randomized controlled trial. J. Reprod. Infant Psychol. 5:9–13.[Medline]
Hebert, L.E., P.A. Scherr, J.L. Bienias, D.A. Bennett, and D.A. Evans. 2003. Alzheimer disease in the US population: prevalence estimates using the 2000 census. Arch. Neurol. 60:1119–1122.
Hong, H.S., E.M. Hwang, H.J. Sim, H.J. Cho, J.H. Boo, S.S. Oh, S.U. Kim, and I. Mook-Jung. 2003. Interferon gamma stimulates beta-secretase expression and sAPPbeta production in astrocytes. Biochem. Biophys. Res. Commun. 307:922–927.[CrossRef][Medline]
Hsiao, K.K., D.R. Borchelt, K. Olson, R. Johannsdottir, C. Kitt, W. Yunis, S. Xu, C. Eckman, S. Younkin, D. Price, et al. 1995. Age-related CNS disorder and early death in transgenic FVB/N mice overexpressing Alzheimer amyloid precursor proteins. Neuron. 15:1203–1218.[CrossRef][Medline]
Hsu, H., J. Xiong, and D.V. Goeddel. 1995. The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation. Cell. 81:495–504.[CrossRef][Medline]
Irizarry, M.C., M. McNamara, K. Fedorchak, K. Hsiao, and B.T. Hyman. 1997. APPSw transgenic mice develop age-related A beta deposits and neuropil abnormalities, but no neuronal loss in CA1. J. Neuropathol. Exp. Neurol. 56:965–973.[Medline]
Itoh, Y., M. Yamada, M. Hayakawa, E. Otomo, and T. Miyatake. 1993. Cerebral amyloid angiopathy: a significant cause of cerebellar as well as lobar cerebral hemorrhage in the elderly. J. Neurol. Sci. 116:135–141.[CrossRef][Medline]
Li, C., and L.B. Hersh. 1995. Neprilysin: assay methods, purification, and characterization. Methods Enzymol. 248:253–263.[CrossRef][Medline]
Li, R., K. Lindholm, L.B. Yang, X. Yue, M. Citron, R. Yan, T. Beach, L. Sue, M. Sabbagh, H. Cai, et al. 2004a. Amyloid beta peptide load is correlated with increased beta-secretase activity in sporadic Alzheimer's disease patients. Proc. Natl. Acad. Sci. USA. 101:3632–3637.
Li, R., L. Yang, K. Lindholm, Y. Konishi, X. Yue, H. Hampel, D. Zhang, and Y. Shen. 2004b. Tumor necrosis factor death receptor signaling cascade is required for amyloid-beta protein-induced neuron death. J. Neurosci. 24:1760–1771.
Matsuoka, Y., M. Picciano, B. Malester, J. LaFrancois, C. Zehr, J.M. Daeschner, J.A. Olschowka, M.I. Fonseca, M.K. O'Banion, A.J. Tenner, et al. 2001. Inflammatory responses to amyloidosis in a transgenic mouse model of Alzheimer's disease. Am. J. Pathol. 158:1345–1354.
Pak, T., P. Cadet, K.J. Mantione, and G.B. Stefano. 2005. Morphine via nitric oxide modulates beta-amyloid metabolism: a novel protective mechanism for Alzheimer's disease. Med. Sci. Monit. 11:BR357–BR366.[Medline]
Patel, N.S., D. Paris, V. Mathura, A.N. Quadros, F.C. Crawford, and M.J. Mullan. 2005. Inflammatory cytokine levels correlate with amyloid load in transgenic mouse models of Alzheimer's disease. J. Neuroinflammation. 2:9.[CrossRef][Medline]
Peschon, J.J., D.S. Torrance, K.L. Stocking, M.B. Glaccum, C. Otten, C.R. Willis, K. Charrier, P.J. Morrissey, C.B. Ware, and K.M. Mohler. 1998. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J. Immunol. 160:943–952.
Pittenger, C., Y.Y. Huang, R.F. Paletzki, R. Bourtchouladze, H. Scanlin, S. Vronskaya, and E.R. Kandel. 2002. Reversible inhibition of CREB/ATF transcription factors in region CA1 of the dorsal hippocampus disrupts hippocampus-dependent spatial memory. Neuron. 34:447–462.[CrossRef][Medline]
Sambamurti, K., R. Kinsey, B. Maloney, Y.W. Ge, and D.K. Lahiri. 2004. Gene structure and organization of the human beta-secretase (BACE) promoter. FASEB J. 18:1034–1036.
Sastre, M., I. Dewachter, S. Rossner, N. Bogdanovic, E. Rosen, P. Borghgraef, B.O. Evert, L. Dumitrescu-Ozimek, D.R. Thal, G. Landreth, et al. 2006. Nonsteroidal anti-inflammatory drugs repress beta-secretase gene promoter activity by the activation of PPARgamma. Proc. Natl. Acad. Sci. USA. 103:443–448.
Saura, C.A., G. Chen, S. Malkani, S.Y. Choi, R.H. Takahashi, D. Zhang, G.K. Gouras, A. Kirkwood, R.G. Morris, and J. Shen. 2005. Conditional inactivation of presenilin 1 prevents amyloid accumulation and temporarily rescues contextual and spatial working memory impairments in amyloid precursor protein transgenic mice. J. Neurosci. 25:6755–6764.
Schmitz, C., B.P. Rutten, A. Pielen, S. Schafer, O. Wirths, G. Tremp, C. Czech, V. Blanchard, G. Multhaup, P. Rezaie, et al. 2004. Hippocampal neuron loss exceeds amyloid plaque load in a transgenic mouse model of Alzheimer's disease. Am. J. Pathol. 164:1495–1502.
Selkoe, D.J. 2003. Aging, amyloid, and Alzheimer's disease: a perspective in honor of Carl Cotman. Neurochem. Res. 28:1705–1713.[CrossRef][Medline]
Shyu, W.C., S.Z. Lin, M.F. Chiang, C.Y. Su, and H. Li. 2006. Intracerebral peripheral blood stem cell (CD34+) implantation induces neuroplasticity by enhancing beta1 integrin-mediated angiogenesis in chronic stroke rats. J. Neurosci. 26:3444–3453.
Simard, A.R., D. Soulet, G. Gowing, J.P. Julien, and S. Rivest. 2006. Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron. 49:489–502.[CrossRef][Medline]
Skalli, O., P. Ropraz, A. Trzeciak, G. Benzonana, D. Gillessen, and G. Gabbiani. 1986. A monoclonal antibody against -smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103:2787–2796.
Sommer, B., and M. Staufenbiel. 1998. A beta peptide deposition in the brains of transgenic mice: evidence for a key event in Alzheimer's disease pathogenesis. Mol. Psychiatry. 3:284–286, 282–283.[CrossRef][Medline]
Song, E.S., M.A. Juliano, L. Juliano, and L.B. Hersh. 2003. Substrate activation of insulin-degrading enzyme (insulysin). A potential target for drug development. J. Biol. Chem. 278:49789–49794.
Sturchler-Pierrat, C., D. Abramowski, M. Duke, K.H. Wiederhold, C. Mistl, S. Rothacher, B. Ledermann, K. Burki, P. Frey, P.A. Paganetti, et al. 1997. Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc. Natl. Acad. Sci. USA. 94:13287–13292.
Tamagno, E., P. Bardini, A. Obbili, A. Vitali, R. Borghi, D. Zaccheo, M.A. Pronzato, O. Danni, M.A. Smith, G. Perry, and M. Tabaton. 2002. Oxidative stress increases expression and activity of BACE in NT2 neurons. Neurobiol. Dis. 10:279–288.[CrossRef][Medline]
Tamagno, E., M. Parola, P. Bardini, A. Piccini, R. Borghi, M. Guglielmotto, G. Santoro, A. Davit, O. Danni, M.A. Smith, et al. 2005. Beta-site APP cleaving enzyme up-regulation induced by 4-hydroxynonenal is mediated by stress-activated protein kinases pathways. J. Neurochem. 92:628–636.[CrossRef][Medline]
Tanzi, R.E., and L. Bertram. 2005. Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective. Cell. 120:545–555.[CrossRef][Medline]
Tarkowski, E., N. Andreasen, A. Tarkowski, and K. Blennow. 2003. Intrathecal inflammation precedes development of Alzheimer's disease. J. Neurol. Neurosurg. Psychiatry. 74:1200–1205.
Tartaglia, L.A., M. Rothe, Y.F. Hu, and D.V. Goeddel. 1993. Tumor necrosis factor's cytotoxic activity is signaled by the p55 TNF receptor. Cell. 73:213–216.[CrossRef][Medline]
Terry, R.D., L.A. Hansen, R. DeTeresa, P. Davies, H. Tobias, and R. Katzman. 1987. Senile dementia of the Alzheimer type without neocortical neurofibrillary tangles. J. Neuropathol. Exp. Neurol. 46:262–268.[Medline]
Thal, D.R., E. Ghebremedhin, M. Orantes, and O.D. Wiestler. 2003. Vascular pathology in Alzheimer disease: correlation of cerebral amyloid angiopathy and arteriosclerosis/lipohyalinosis with cognitive decline. J. Neuropathol. Exp. Neurol. 62:1287–1301.[Medline]
Thifault, S., R. Lalonde, N. Sanon, and P. Hamet. 2002. Comparisons between C57BL/6J and A/J mice in motor activity and coordination, hole-poking, and spatial learning. Brain Res. Bull. 58:213–218.[CrossRef][Medline]
Tian, J., J. Shi, K. Bailey, and D.M. Mann. 2003. Negative association between amyloid plaques and cerebral amyloid angiopathy in Alzheimer's disease. Neurosci. Lett. 352:137–140.[CrossRef][Medline]
Tiraboschi, P., M.N. Sabbagh, L.A. Hansen, D.P. Salmon, A. Merdes, A. Gamst, E. Masliah, M. Alford, L.J. Thal, and J. Corey-Bloom. 2004. Alzheimer disease without neocortical neurofibrillary tangles: "a second look." Neurology. 62:1141–1147.
Tobe, M., Y. Isobe, H. Tomizawa, T. Nagasaki, H. Takahashi, T. Fukazawa, and H. Hayashi. 2003. Discovery of quinazolines as a novel structural class of potent inhibitors of NF-kappa B activation. Bioorg. Med. Chem. 11:383–391.[CrossRef][Medline]
Van Dam, D., R. D'Hooge, M. Staufenbiel, C. Van Ginneken, F. Van Meir, and P.P. De Deyn. 2003. Age-dependent cognitive decline in the APP23 model precedes amyloid deposition. Eur. J. Neurosci. 17:388–396.[CrossRef][Medline]
Van Dooren, T., D. Muyllaert, P. Borghgraef, A. Cresens, H. Devijver, I. Van der Auwera, S. Wera, I. Dewachter, and F. Van Leuven. 2006. Neuronal or glial expression of human apolipoprotein e4 affects parenchymal and vascular amyloid pathology differentially in different brain regions of double- and triple-transgenic mice. Am. J. Pathol. 168:245–260.
Vassar, R., B.D. Bennett, S. Babu-Khan, S. Kahn, E.A. Mendiaz, P. Denis, D.B. Teplow, S. Ross, P. Amarante, R. Loeloff, et al. 1999. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 286:735–741.
Vinters, H.V. 1987. Cerebral amyloid angiopathy. A critical review. Stroke. 18:311–324.
Wang, H., G.D. Ferguson, V.V. Pineda, P.E. Cundiff, and D.R. Storm. 2004. Overexpression of type-1 adenylyl cyclase in mouse forebrain enhances recognition memory and LTP. Nat. Neurosci. 7:635–642.[CrossRef][Medline]
Weiner, H.L., and D.J. Selkoe. 2002. Inflammation and therapeutic vaccination in CNS diseases. Nature. 420:879–884.[CrossRef][Medline]
Wilcock, D.M., G. DiCarlo, D. Henderson, J. Jackson, K. Clarke, K.E. Ugen, M.N. Gordon, and D. Morgan. 2003. Intracranially administered anti-Abeta antibodies reduce beta-amyloid deposition by mechanisms both independent of and associated with microglial activation. J. Neurosci. 23:3745–3751.
Wilcock, D.M., A. Rojiani, A. Rosenthal, G. Levkowitz, S. Subbarao, J. Alamed, D. Wilson, N. Wilson, M.J. Freeman, M.N. Gordon, and D. Morgan. 2004. Passive amyloid immunotherapy clears amyloid and transiently activates microglia in a transgenic mouse model of amyloid deposition. J. Neurosci. 24:6144–6151.
Wiltfang, J., A. Smirnov, B. Schnierstein, G. Kelemen, U. Matthies, H.W. Klafki, M. Staufenbiel, G. Huther, E. Ruther, and J. Kornhuber. 1997. Improved electrophoretic separation and immunoblotting of beta-amyloid (A beta) peptides 1-40, 1-42, and 1-43. Electrophoresis. 18:527–532.[CrossRef][Medline]
Winkler, D.T., L. Bondolfi, M.C. Herzig, L. Jann, M.E. Calhoun, K.H. Wiederhold, M. Tolnay, M. Staufenbiel, and M. Jucker. 2001. Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy. J. Neurosci. 21:1619–1627.
Yan, Q., J. Zhang, H. Liu, S. Babu-Khan, R. Vassar, A.L. Biere, M. Citron, and G. Landreth. 2003. Anti-inflammatory drug therapy alters beta-amyloid processing and deposition in an animal model of Alzheimer's disease. J. Neurosci. 23:7504–7509.
Yang, L., K. Lindholm, Y. Konishi, R. Li, and Y. Shen. 2002. Target depletion of distinct tumor necrosis factor receptor subtypes reveals hippocampal neuron death and survival through different signal transduction pathways. J. Neurosci. 22:3025–3032.
Yang, L.B., K. Lindholm, R. Yan, M. Citron, W. Xia, X.L. Yang, T. Beach, L. Sue, P. Wong, D. Price, et al. 2003. Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nat. Med. 9:3–4.[CrossRef][Medline]
Zhou, J., M.I. Fonseca, R. Kayed, I. Hernandez, S.D. Webster, O. Yazan, D.H. Cribbs, C.G. Glabe, and A.J. Tenner. 2005. Novel Abeta peptide immunogens modulate plaque pathology and inflammation in a murine model of Alzheimer's disease. J. Neuroinflammation. 2:28.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
Related Article
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
