Reduction of Amyloid Angiopathy and Aβ Plaque Burden after Enriched Housing in TgCRND8 Mice: Involvement of Multiple Pathways

Abstract

Diversity and intensity of intellectual and physical activities seem to have an inverse relationship with the extent of cognitive decline in Alzheimer's disease (AD). To study the interaction between an active lifestyle and AD pathology, female TgCRND8 mice carrying human APPswe+ind were transferred into enriched housing. Four months of continuous and diversified environmental stimulation resulted in a significant reduction of β-amyloid (Aβ) plaques and in a lower extent of amyloid angiopathy. Neither human amyloid precursor protein (APP) mRNA/protein levels nor the level of carboxy-terminal fragments of APP nor soluble Aβ content differed between both groups, making alterations in APP expression or processing unlikely as a cause of reduced Aβ deposition. Moreover, DNA microarray analysis revealed simultaneous down-regulation of proinflammatory genes as well as up-regulation of molecules involved in anti-inflammatory processes, proteasomal degradation, and cholesterol binding, possibly explaining reduced Aβ burden by lower aggregation and enhanced clearance of Aβ. Additionally, immunoblotting against F4/80 antigen and morphometric analysis of microglia (Mac-3) revealed significantly elevated microgliosis in the enriched brains, which suggests increased amyloid phagocytosis. In summary, this study demonstrates that the environment interacts with AD pathology at dif-ferent levels. Diversity and intensity of intellectual and physical activities seem to have an inverse relationship with the extent of cognitive decline in Alzheimer's disease (AD). To study the interaction between an active lifestyle and AD pathology, female TgCRND8 mice carrying human APPswe+ind were transferred into enriched housing. Four months of continuous and diversified environmental stimulation resulted in a significant reduction of β-amyloid (Aβ) plaques and in a lower extent of amyloid angiopathy. Neither human amyloid precursor protein (APP) mRNA/protein levels nor the level of carboxy-terminal fragments of APP nor soluble Aβ content differed between both groups, making alterations in APP expression or processing unlikely as a cause of reduced Aβ deposition. Moreover, DNA microarray analysis revealed simultaneous down-regulation of proinflammatory genes as well as up-regulation of molecules involved in anti-inflammatory processes, proteasomal degradation, and cholesterol binding, possibly explaining reduced Aβ burden by lower aggregation and enhanced clearance of Aβ. Additionally, immunoblotting against F4/80 antigen and morphometric analysis of microglia (Mac-3) revealed significantly elevated microgliosis in the enriched brains, which suggests increased amyloid phagocytosis. In summary, this study demonstrates that the environment interacts with AD pathology at dif-ferent levels. Alzheimer's disease (AD) is the most prevalent form of senile dementia worldwide. It is characterized by two major histological hallmarks: senile plaques, ie, extracellular deposits mainly consisting of β-amyloid (Aβ), and neurofibrillary tangles, ie, intracellular accumulations of hyperphosphorylated tau protein.1Mattson M Pathways towards and away from Alzheimer's disease.Nature. 2004; 430: 631-639Crossref PubMed Scopus (2430) Google Scholar AD patients show progressing cognitive decline as well as noncognitive behavioral symptoms such as wandering, sleep disturbance, and physical aggression.2Finkel SI Behavioral and psychological symptoms of dementia.Clin Geriatr Med. 2003; 19: 799-824Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar There are various risk factors for AD including age, family history, or apolipoprotein E ε4 genotype.3Ritchie K Lovestone S The dementias.Lancet. 2002; 360: 1759-1766Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar Epidemiological studies additionally suggest that the amount of time spent on intellectual and physical activities negatively correlates with the extent of cognitive decline and even risk of developing AD.4Friedland RP Fritsch T Smyth KA Koss E Lerner AJ Chen CH Petot GJ Debanne SM Patients with Alzheimer's disease have reduced activities in midlife compared with healthy control-group members.Proc Natl Acad Sci USA. 2001; 98: 3440-3445Crossref PubMed Scopus (359) Google Scholar, 5Wilson RS Mendes De Leon CF Barnes LL Schneider JA Bienias JL Evans DA Bennett DA Participation in cognitively stimulating activities and risk of incident Alzheimer disease.JAMA. 2002; 287: 742-748Crossref PubMed Scopus (970) Google Scholar Although it cannot be excluded that lower activity levels are early subclinical symptoms, one should consider them also as a risk factor. In line with this assumption is the use of cognitive training as a rehabilitative measure resulting in deceleration of dementia progress. However, the underlying molecular pathways are essentially unknown.In laboratory rodents cognitive, physical, and social stimulation can be regulated by altering housing conditions. It is well established that living in an enriched environment provided by additional structural or social stimuli may increase locomotor and exploratory activity, improve learning and memory performance, increase dendritic sprouting and synapse formation in the neocortex and hippocampus as well as neurogenesis in the dentate gyrus,6van Praag H Kempermann G Gage FH Neural consequences of environmental enrichment.Nat Rev Neurosci. 2000; 1: 191-198Crossref PubMed Scopus (1904) Google Scholar and affects behavioral, endocrinological, and immunological parameters.7Marashi V Barnekow A Ossendorf E Sachser N Effects of different forms of environmental enrichment on behavioral, endocrinological, and immunological parameters in male mice.Horm Behav. 2003; 43: 281-292Crossref PubMed Scopus (185) Google Scholar Environmental enrichment also facilitates recovery from acute brain lesions, again accompanied by structural changes such as increased dendritic branching and spine density,8Johansson BB Belichenko PV Neuronal plasticity and dendritic spines: effect of environmental enrichment on intact and postischemic rat brain.J Cereb Blood Flow Metab. 2002; 22: 89-96Crossref PubMed Scopus (216) Google Scholar tightly controlled by a complex concert of a variety of genes and proteins.9Keyvani K Sachser N Witte OW Paulus W Gene expression profiling in the intact and injured brain following environmental enrichment.J Neuropathol Exp Neurol. 2004; 63: 598-609PubMed Google Scholar Studies regarding the effect of enriched housing (EH) on animal models of neurodegenerative diseases have demonstrated that EH delays disease progression in a mouse model of Huntington's disease10van Dellen A Blakemore C Deacon R York D Hannan AJ Delaying the onset of Huntington's in mice.Nature. 2000; 404: 721-722Crossref PubMed Scopus (412) Google Scholar or protects mice from pharmacologically induced Parkinsonism.11Faherty CJ Raviie Shepherd K Herasimtschuk A Smeyne RJ Environmental enrichment in adulthood eliminates neuronal death in experimental Parkinsonism.Brain Res Mol Brain Res. 2005; 134: 170-179Crossref PubMed Scopus (197) Google Scholar Concerning AD, a few studies on the effects of environmental stimulation produced partly contradictory results. Taken together, these studies strongly suggest that EH affects both cognitive abilities12Arendash G Garcia M Costa D Cracchiolo J Wefes I Potter H Environmental enrichment improves cognition in aged Alzheimer's transgenic mice despite stable beta-amyloid deposition.Neuroreport. 2004; 15: 1751-1754Crossref PubMed Scopus (183) Google Scholar, 13Jankowsky JL Melnikova T Fadale DJ Xu GM Slunt HH Gonzales V Younkin LH Younkin SG Borchelt DR Savonenko AV Environmental enrichment mitigates cognitive deficits in a mouse model of Alzheimer's disease.J Neurosci. 2005; 25: 5217-5224Crossref PubMed Scopus (403) Google Scholar and the development of an AD-like pathology13Jankowsky JL Melnikova T Fadale DJ Xu GM Slunt HH Gonzales V Younkin LH Younkin SG Borchelt DR Savonenko AV Environmental enrichment mitigates cognitive deficits in a mouse model of Alzheimer's disease.J Neurosci. 2005; 25: 5217-5224Crossref PubMed Scopus (403) Google Scholar, 14Jankowsky JL Xu G Fromholt D Gonzales V Borchelt DR Environmental enrichment exacerbates amyloid plaque formation in a transgenic mouse model of Alzheimer disease.J Neuropathol Exp Neurol. 2003; 62: 1220-1227Crossref PubMed Scopus (171) Google Scholar, 15Lazarov O Robinson J Tang YP Hairston IS Korade-Mirnics Z Lee VMY Hersh LB Sapolsky RM Mirnics K Sisodia SS Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice.Cell. 2005; 120: 701-713Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar, 16Adlard PA Perreau VM Pop V Cotman CW Voluntary exercise decreases amyloid load in a transgenic model of Alzheimer's disease.J Neurosci. 2005; 25: 4217-4221Crossref PubMed Scopus (598) Google Scholar in AD mouse models, although reasons for discrepant results and involved mechanisms have remained unclear. We kept female TgCRND8 mice under standard housing (SH) and EH conditions from day 30 until 5 months of age to gain insight into mechanisms underlying environmentally evoked effects on Aβ pathology. Compared to other murine models of AD, TgCRND8 mice exhibit Aβ plaques very early (∼3 months), accompanied by Aβ deposition in vessel walls, astrogliosis/microgliosis, and cognitive deficits,17Chishti MA Yang DS Janus C Phinney AL Horne P Pearson J Strome R Zuker N Loukides J French J Turner S Lozza G Grilli M Kunicki S Morissette C Paquette J Gervais F Bergeron C Fraser PE Carlson GA St. George-Hyslop P Westaway D Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695.J Biol Chem. 2001; 276: 21562-21570Crossref PubMed Scopus (766) Google Scholar, 18Janus C Pearson J McLaurin J Mathews PM Jiang Y Schmidt SD Chishti MA Horne P Heslin D French J Mount HT Nixon RA Mercken M Bergeron C Fraser PE St. George-Hyslop P Westaway D Aβ peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease.Nature. 2000; 408: 979-982Crossref PubMed Scopus (1362) Google Scholar which are typical symptoms associated with AD.Materials and MethodsAnimals and Housing ConditionsWe investigated 18 female transgenic mice of the TgCRND8 line that carries a double-mutated form of the human amyloid precursor protein 695 (APP695), the Swedish and Indiana mutations, under control of the Syrian hamster prion promoter, on a hybrid C57BL/6-C3H/HeJ background.17Chishti MA Yang DS Janus C Phinney AL Horne P Pearson J Strome R Zuker N Loukides J French J Turner S Lozza G Grilli M Kunicki S Morissette C Paquette J Gervais F Bergeron C Fraser PE Carlson GA St. George-Hyslop P Westaway D Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695.J Biol Chem. 2001; 276: 21562-21570Crossref PubMed Scopus (766) Google Scholar, 18Janus C Pearson J McLaurin J Mathews PM Jiang Y Schmidt SD Chishti MA Horne P Heslin D French J Mount HT Nixon RA Mercken M Bergeron C Fraser PE St. George-Hyslop P Westaway D Aβ peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease.Nature. 2000; 408: 979-982Crossref PubMed Scopus (1362) Google Scholar At 30 days of age, animals were transferred to the experimental housing conditions. Nine transgenic mice were housed (together with wild-type littermates that were not further analyzed for the present study) in groups of three or four in SH conditions, nine transgenics were housed in equally composed groups in EH conditions (at least one animal of each genotype per cage). SH consisted of transparent polycarbonate cages (38 cm × 22 cm × 15 cm) with sawdust as bedding material. Enriched cages contained further nesting material, a plastic inset, and a wooden scaffolding. In addition, EH animals had access to a second, so-called stimulus cage during the dark phase that was connected to the home cage by a Plexiglas tunnel. The stimulus cage contained different stimulus objects divided in five categories: 1) permanently, a sisal rope and gnawing wood were available. In addition, one object of the categories 2) tunnels, 3) balls, 4) soft materials, and 5) varied locomotive substrates including wooden ramps and ladders, plastic stairs, as well as runningwheels were inside. Every day, one stimulus object of a daily switching category was exchanged to expose EH mice to novel environmental stimulation. A photoperiod of a 12-hour light/dark cycle was maintained. All experimental procedures were in accordance with the guidelines of the local animal care commission.Brain Tissue PreparationMice were decapitated at 150 days of age. Brains were removed and one hemisphere was fixed in 4% buffered formaldehyde for 24 hours followed by dehydration and paraffin embedding. The other hemisphere was immediately snap-frozen in liquid nitrogen. Total RNA and subsequently protein were extracted from the same homogenized tissue of the whole cerebral hemisphere (without cerebellum and brain stem) using TRIzol reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer's instructions. RNA was DNase-treated and cleaned. RNA quality was assessed by Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Palo Alto, CA). Proteins were dissolved in 1% sodium dodecyl sulfate. Because of loss of one pellet, the number of SH mice was eight for RNA and protein analyses.ImmunohistochemistryFor Aβ staining three pairs of 2-μm sagittal brain sections of each transgenic animal were pretreated with formic acid and automatically stained in a TechMate instrument (DAKO, Hamburg, Germany) with 6F/3D anti-Aβ monoclonal antibody to residues 8 to 17 (1:100, DAKO) followed by the DAKO StreptABC complex-horseradish peroxidase conjugated Duet anti-mouse/rabbit antibody kit and development with 3,3′-diaminobenzidine. For Mac-3 staining two pairs of slices were pretreated with ethylenediaminetetraacetic acid buffer (pH 8.5) in a vegetable steamer for 30 minutes. Primary anti-Mac-3 antibody (1:100, BD Biosciences, Heidelberg, Germany) was incubated overnight at 4°C and developed using the biotin/avidin technique. Counterstaining was performed with hematoxylin. The pairs of sections (10 μm distance) were situated between 100 and 300 μm lateral from the mid-sagittal fissure. Each staining was performed in two consecutive procedures making sure that brains of both experimental groups were equally distributed in all procedures.Quantitative Evaluation of Aβ and Mac-3 ImmunoreactivityTo quantify Aβ plaque burden, neocortices and hippocampi of all stained sections were digitized (Olympus BX50, ColorView II, charge-coupled device camera; Olympus, Hamburg, Germany) under constant light and filter settings. Color images were converted to grayscale by extracting blue to gray values to obtain best contrast between positive immunoreactivity and background. A constant threshold was chosen for all images to detect immunoreactive staining (analySIS 5; Soft Imaging System, Münster, Germany). Plaque number, size, and total area were determined in total neocortex and hippocampus. Absolute values of plaque burden were related to the investigated area.The severity of amyloid angiopathy was assessed semiquantitatively in the whole cerebral hemisphere by light microscopy at ×400 magnification. Leptomeningeal and intracerebral blood vessels with a visible lumen and being at least 0.01 mm in diameter were considered positive when Aβ immunoreactivity was present in a circumferential or patchy pattern within the vessel walls. The percentage of Aβ-positive vessels was acquired in relation to total counted vessels. The observer was blind to experimental condition.The total number of Mac-3-positive microglia/macrophages was counted in the whole neocortical and hippocampal area using ×200 magnification with a morphometrical lattice. In average 15 ± 2 optical fields per slice were examined. The number of microglial cells is given as cells per mm2. Microglia were counted by two raters who were blind to experimental condition.Protein AnalysisProtein concentration was assessed by the DC Protein Assay (Bio-Rad, Munich, Germany). Aβ1-40 and Aβ1-42 peptide levels of each animal were determined using enzyme-linked immunosorbent assay (ELISA) (Biosource, Solingen, Germany). For Western blot analysis of Aβ1-40 and Aβ1-42, samples were subjected to sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis as previously reported.19Klafki HW Wiltfang J Staufenbiel M Electrophoretic separation of betaA4 peptides (1-40) and (1-42).Anal Biochem. 1996; 237: 24-29Crossref PubMed Scopus (98) Google Scholar Tris/Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis of full-length APP and C-terminal fragments was performed as described by Schägger and von Jagow.20Schägger H von Jagow G Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.Anal Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10442) Google Scholar Subsequently proteins were electrophoretically transferred onto nitrocellulose, and Western blot was performed with the antibodies 6E10 (1:1000, directed against Aβ1-16; Signet Laboratories, Dedham, MA), anti-APP C-terminal (no. 171610, 1:10,000, raised against the C-terminal 20 residues of human APP; Calbiochem/EMD Biosciences, Darmstadt, Germany), or anti-α-tubulin CP06 (1:1000, Calbiochem), followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham, Freiburg, Germany). Detection was done by the use of ECL-Advance (Amersham), band intensity was analyzed with QuantityOne software (Bio-Rad, Milan, Italy).F4/80 antigen and glial fibrillary acidic protein (GFAP) levels were also assessed by Western blot analysis. Protein (20 or 5 μg) of each animal was loaded on a 7.5% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel for F4/80 and GFAP, respectively. After electrophoresis and wet blotting, membranes were blocked with 5% nonfat milk in phosphate-buffered saline buffer (containing 0.5% Tween 20) for F4/80 Western blot or in TST buffer (10 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 0.05% Tween 20) for GFAP Western blot and incubated for 1 hour at room temperature with F4/80 antibody (MCAP497, 1:500; Serotec, Düsseldorf, Germany) or overnight at 4°C with GFAP antibody (Z0334, 1:7500; DAKO) followed by secondary antibody [401416, 1:5000 (Calbiochem, San Diego, CA) for F4/80; A2074, 1:15000 (Sigma-Aldrich, Munich, Germany) for GFAP] and peroxidase-catalyzed enhanced chemiluminescence (ECL-Plus; Amersham Biosciences, Freiburg, Germany). β-Actin was used for normalization (primary and secondary antibody 1:10,000 each; Sigma-Aldrich). Protein expression levels were determined by a densitometry software Gel-Pro analyzer (Media Cybernetics, Silver Spring, MD). All samples were analyzed in duplicate.Corticosterone AssayTrunk blood was collected from each animal in heparinized capillaries directly after decapitation (within 3 minutes from disturbing the animals' cage). Plasma corticosterone concentrations were determined in duplicate by radioimmunoassay without chromatography using corticosterone antiserum (C8784, Sigma-Aldrich) with the following cross-reactivity: progesterone, 15.7%; 11-deoxycorticosterone, 20%; 20α-hydroxyprogesterone, 8.8%; cortisol, 4.5%; 20β-hydroxyprogesterone, 5.2%; testosterone, 7.9%; 17-hydroxyprogesterone, 1.8%; androstenedione, 2.6%; aldosterone, 4.4%; 11-deoxycortisol, 1.3%; 5α-dihydrotestosterone, 1.4%; cortisone, 3.2%; and androsterone, dehydroepiandrosterone, estrone, 17β-estradiol, estriol, <0.1%. All samples were run in a single assay. The intra-assay coefficient of variation was <4%. Further details of assay performance have been described elsewhere.2121. Kaiser S, Kirtzeck M, Hornschuh G, Sachser N: Sex-specific difference in social support—a study in female guinea pigs. Physiol Behav 79:297–303Google ScholarDNA MicroarrayIsolated RNA from animals of the same housing condition was pooled, receiving four pools consisting of two biological duplicates termed standard 1 (four animals), standard 2 (four animals), enriched 1 (four animals), and enriched 2 (five animals), each pool containing 20 μg of RNA. Mouse genome 430A 2.0 array hybridization was performed according to the manufacturer's instructions (Affymetrix, Wooburn Green, United Kingdom).Raw expression data were accumulated by GeneChip Operating System software (Gecos v1.2; Affymetrix). CEL-files were then imported to the software package CoBi/Expressionist Pro 1.0 (GeneData, Basel, Switzerland). Before analysis, signal values were normalized to the logarithmic mean with a reference value of 1000 to ensure comparability. Expressionist's internal quality control was set to P = 0.05. The mean expression values received from biological duplicates in EH versus SH groups were used to accomplish pair-wise comparison and assess fold changes. Only genes with a mean expression value greater than 100 in one of the two groups and a 1.5-fold differential expression between the groups were used for further investigations. Differences of P < 0.05 (Student's t-test) were considered significant. Genes showing significant different regulation between the two experimental conditions were categorized according to their biological function by means of Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA) and via literature survey (PubMed).Quantitative Real-Time PCR (TaqMan Assay)To assess human APP transgene expression levels, cDNA was synthesized from 2 μg of RNA of each animal using Omniscript RT kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. To verify expression levels of the microarray experiment, RNA was pooled before reverse transcription corresponding to the four pools used for the microarray analysis. polymerase chain reaction (PCR) primers and TaqMan probes were designed using Primer Express software (version 2.0, Applied Biosystems). Specificity of each amplicon was controlled by BLAST search. GAPDH was used for normalization. All assays were run in triplicate. Primers and cycling conditions are available on request.Statistical AnalysisNormal distribution of all data sets was confirmed by one-sample Kolmogorov-Smirnov test. As all data were normally distributed, SH and EH groups were compared using an unpaired t-test. All tests were applied two-tailed except for the morphometrical analysis of microglia (as the confirmation for Western blot data) using the software package SPSS (version 12.0.1). When necessary, Bonferroni correction was calculated for multiple testing. Differences were considered significant at P < 0.05.ResultsReduced Aβ Deposition in EH MiceWe first investigated the effect of EH on Aβ deposition by immunohistochemical staining and digital image analysis (Figure 1, A and B). The number of Aβ plaques in the neocortex and hippocampus together was significantly reduced (by 28.8%, P′ = 0.024, P′: Bonferroni corrected) in EH animals compared to SH mice (Figure 1C). In addition, there was a 47.3% reduction in the total Aβ-positive area (Figure 1D, P′ = 0.022). The mean size of plaques was 25% smaller in EH mice (P′ = 0.024). In neocortex alone, plaque number was significantly reduced by 29.2% (P′ = 0.026), Aβ-positive area by 45.3% (P′ = 0.020) and the mean size of plaques by 24.3% (P′ = 0.016). In hippocampus, there was only a slight but statistically not significant reduction in plaque number by 19.6% (P′ = 0.308), Aβ-positive area (by 42.9%, P′ = 0.122) and mean plaque size (by 28.7%, P′ = 0.134). The extent of amyloid angiopathy was also significantly reduced in EH mice. The percentage of amyloid laden leptomeningeal vessels decreased by 30% (P = 0.020), and that of intracerebral vessels by 59% (Figure 1E, P = 0.011). Paraffin sections of two wild-type mice were stained as control. None of these animals showed any Aβ deposits.Soluble Aβ Levels as Well as APP Expression and Processing Did Not Differ Between Housing ConditionsThere was no significant difference in the levels of soluble Aβ1-40 or Aβ1-42 between EH and SH mice as measured by ELISA (Figure 2, A and B; P = 0.853 and P = 0.359, respectively). The relation between Aβ1-40 and Aβ1-42 levels did not differ between the two housing conditions (P = 0.743). Similar results could be obtained by Western blot analysis (Figure 2, C–E). There were again no differences between EH and SH mice in Aβ1-40 (Figure 2C, P = 0.691), Aβ1-42 levels (Figure 2D, P = 0.858), as well as in the relation between Aβ1-40 and Aβ1-42 (P = 0.333).Figure 2APP expression and processing. A–D: Steady state levels of soluble Aβ1-40 and Aβ1-42 as measured by ELISA and Western blot. E: Representative Western blot with antibody specific for Aβ (6E10). There are slight variations in levels of detergent-soluble Aβ1-40 and Aβ1-42, but these are fully consistent with the variations observed with a protein loading control, α-tubulin. F: Representative Western blot data showing no differences in full-length APP and in proteolytic fragments of APP (CTFβ). A faint CTFα fragment was only detectable after a longer exposition duration (F*). G–I: Steady state levels of APP mRNA and protein as well as CTFβ stub. No differences were seen in EH versus SH mice. Data are given as means ± SD; statistics, t-test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To assess the expression levels of human APP transgene as a possible reason of altered plaque burden, we performed quantitative real-time reverse transcriptase (RT)-PCR as well as Western blot analysis against APP (Figure 2F, top row). No differences between EH and SH mice could be detected, neither at transcriptional nor translational levels (Figure 2, G and H; P = 0.452 and P = 0.457, respectively). Additionally, the level of the proteolytic fragment APP-CTFβ was measured to evaluate whether alteration in APP processing could account for reduction of Aβ deposition (Figure 2F, bottom row). Again, there was no significant difference in CTFβ levels between EH and SH mice (Figure 2I, P = 0.818). A weak CTFα band was detectable after a longer exposition duration (Figure 2F*). A densitometric quantification of this band was hampered because of its faintness, although it appeared virtually unchanged in all examined animals.Housing Conditions Did Not Influence Glucocorticoid LevelsCorticosterone as main glucocorticoid in mice was measured to determine the stress level of the animals. Plasma corticosterone levels did also not differ between EH and SH mice (P = 0.567).Enhanced Microgliosis in EH MiceTo determine the extent of activated microglia and astroglia, the expression of cell-type-specific markers was assessed by Western blot analysis. Levels of the astrocytic marker GFAP did not differ between brains from EH and SH conditions (Figure 3A, P = 0.260). In contrast, levels of F4/80 antigen, a 160-kd glycoprotein specifically expressed in microglia,22Austyn JM Gordon S F4/80, a monoclonal antibody directed specifically against the mouse macrophage.Eur J Immunol. 1981; 11: 805-815Crossref PubMed Scopus (1279) Google Scholar were significantly elevated by 118% in EH brains (Figure 3B, P = 0.041). To confirm this result, we performed morphometrical analysis using immunohistochemical staining against Mac-3. EH mice showed an enhancement of 30% in number of activated microglia per mm2 (Figure 3, C and D; P = 0.032). Interestingly, we could very often observe that ramifications of microglial cells appear to infiltrate the core of amyloid plaques (Figure 3D).Figure 3A and B: Levels of GFAP and F4/80 antigen in SH and EH mice as measured by Western blot analysis. In contrast to astrocytic marker GFAP, F4/80 antigen was significantly elevated in EH when compared with SH mice. C: Additionally, the number of microglial cells per mm2 that were counted after immunohistochemistry against Mac-3 was also increased in EH mice. D: Representative picture of Mac-3 immunostaining (P, plaque) showing ring formation of ramified and elongated microglial cells surrounding and infiltrating the amyloid plaques. Data are given as means ± SD; statistics, t-test. Original magnifications: ×200; ×400 (inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Microarray Analysis Revealed Multiple Pathways that May Be Involved in Aβ Burden ReductionExpression levels of the housekeeping genes GAPDH and β-actin showed no significant differences between the four arrays (range: 1.005-fold to 1.01-fold). Two hundred thirty genes revealed different expression values between the two experimental conditions (P < 0.05). Two hundred twenty-one of these genes could be categorized into 14 groups concerning the main function of the protein products in a biological context based on Ingenuity Pathway Analysis and literature survey (PubMed; Figure 4). Nine genes remained unclassified. The entire list of all 230 genes can be found as Supplementary Table 1 (see http://ajp.amjpathol.org).Figure 4Number of genes categorized into different classes concerning their biological function based on Ingenuity Pathway Analysis and literature survey.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For explaining reduced pl