Sastre et al. 10.1073/pnas.0503839103. |
Fig. 5. (A) Quantification of Ab levels (n = 5) in N2a-sw cells transfected with PPARg2 wild-type or mutated PPARg2-E499Q stimulated with IFN-g (1 ng/ml) + TNF-a (30 ng/ml) overnight and then incubated with ibuprofen (IBU) (10 mM) for 4 h. Columns represent mean ± SEM, control (white), IFN-g (1 ng/ml) + TNF-a (30 ng/ml) (black), IFN-g (1 ng/ml) + TNF-a (30 ng/ml) + ibuprofen (hatched). Asterisks, significant differences between control and treatment; #, significant differences between treatment with cytokines alone and with NSAIDs. *, P £ 0.05; **, P £ 0.01; ***, P £ 0.001; #, P £ 0.05; ##, P £ 0.01; ###, P £ 0.001; ANOVA followed by a Tukey post-hoc test. (B) PPARg activity was measured as activation of a PPRE luciferase reporter construct in N2a-sw cells (n = 3). Cells were stimulated as above. Asterisks, significant differences between control and treatment. *, P £ 0.05; **, P £ 0.01; ***, P £ 0.001. ANOVA followed by a Tukey post-hoc test.
Fig. 6. (A) Schematic representation of the deletion mutants of BACE1 promoter. It also indicates the site for the potential PPRE. (B) Activity of BACE1 promoter deletion mutants in PPARg wild-type, knockout, and knockout mouse embryonic fibroblasts (MEF) transfected with PPARg cDNA. The highest luciferase activity was observed in the BPR-Del mutant (–1 to –753 bp). The BPR-Nco deletion mutant contained –1 to –514 bp. The promoter activity of the mutant BPR-Avr (–1 to –417) was significantly reduced. n = 5. (C) The same deletion mutants were transfected in N2a-sw cells, incubated with IFN-g (1 ng/ml) + TNF-a (30 ng/ml) or ibuprofen (10 mM),
Fig. 7. (A) Double immunostaining of PPARg and GFAP or NeuN in sections from human AD brain. PPARg immunoreactivity is present in astrocytes and neurons, mainly perinuclear. (B) Gel-shift analysis of the binding of a oligonucleotide probe for BACE1-PPRE using nuclear extracts from brains of control and AD patients. (C) Quantification of the binding of brain nuclear extracts to the BACE1-PPRE probe from five controls and five AD patients. Columns represent mean ± SEM, n = 4. Asterisks, significant differences between control and AD patients. *, P £ 0.05; **, P £ 0.01; ***, P £ 0.001. Student’s t test.
Fig. 8. Modulation of Ab generation by neuroinflammatory mechanisms. Amyloid peptides cause microglial activation and astrocytosis and therefore increased secretion of specified cytokines, which will suppress PPARg expression and subsequently increase BACE1 transcription and Ab generation, initiating the whole process again. This vicious circle can be modulated at PPARg level, by some nonsteroidal antiinflammatory drugs (NSAIDs) and thiazolidines, which prevent the decrease of PPARg transcription by inflammatory mediators and increase the binding of PPARg to the PPRE domain in BACE1.
Supporting Text
Because peroxisome proliferator-activated receptor g (PPARg) transcriptional activation depends on ligand binding, we determined whether transfection of N2a-APPsw cells with a mutant PPARg affected the production of amyloid b (Ab). PPARg2-E499Q carries a mutation in the AF2 region of the receptor and does not show ligand-dependent transcriptional activation (1). The N2a-APPsw cells transfected with PPARg2-E499Q did not result in any change in Ab secretion following stimulation by proinflammatory cytokines or by incubation with ibuprofen, relative to N2a-APPsw cells transfected with wild-type PPARg2 (Fig. 5A).
Moreover, to define the capability of PPARg to modulate transcriptional transactivation, we examined the activity of a PPAR responsive element (PPRE) reporter construct, transiently transfected in N2a cells. Incubation with TNF-a + IFN-g decreased the luciferase activity, and this effect was abolished by coincubation with indomethacin and ibuprofen (Fig. 5B).
b-site APP cleaving enzyme (BACE1) mRNA levels transcription of N2a cells were not increased when actinomycine D (5 mg/ml) was added before incubation with TNF-a + IFN-g, indicating that inflammation led to de novo synthesis of BACE1 mRNA (data not shown).
To define the elements in the BACE1 gene promoter that are critical for the PPARg repression, we analyzed truncated constructs by removing sequence segments that appeared potentially important for the regulation of the BACE1 gene (Fig. 6A). The promoter activity of BPR.Del (–1 to –753) was significantly higher than that of the original 1.5-kb promoter construct, suggesting suppressor elements to be located between –754 and –1,541 bp (Fig. 6A) (2). The activity of this construct was high when tested in cells with or without PPARg (Fig. 6B), indicating that the deleted region could contain consensus-binding sites for PPARg or other transcription factors modulated by PPARg. On the other hand, the BPR-Nco (–1 to –514) and -BPR-Avr (-1 to -417) deletion constructs were less active, suggesting the presence of activators of BACE1 transcription between –515 and –753 (2). These deletion mutants showed low BACE1 activity independent of the presence of PPARg in the MEF cells, implying that no PPARg-dependent repression sites are localized in this region (Fig. 6B).
We further examined the activity of the BACE1 gene promoter deletion constructs also in neuroblastoma N2a cells. Incubation with TNF-a + IFN-g or ibuprofen did not modify the activity of the mutants (Fig. 6C).
To determine whether PPARg is important for BACE1 regulation in humans, we examined the levels of expression of PPARg in postmortem brain sections from Alzheimer’s disease (AD) patients. The immunohistochemical assessment of frontal cortex indicated that PPARg is expressed in astrocytes and neurons, respectively revealed by costaining with antibodies to GFAP and NeuN (Fig. 7A). PPARg was strongly expressed in perinuclear location, in agreement with their function as a nuclear receptor.
To investigate whether the binding of PPARg to the BACE1 gene promoter was affected, we performed gel-shift analysis with the PPREwt probe after incubation with nuclear extracts from brain of five AD patients and five controls. The retarded band corresponding to the PPRE-complex was reduced by 50% (Fig. 7 B and C), indicating that PPARg protein levels and its binding are decreased in AD brain. Combined with all of the other data, these findings strongly point to a direct role of PPARg in the increased BACE1 transcription and activity in AD, facilitating the generation of Ab.
Methods
Brain frozen sections from an AD patient were fixed with 4% paraformaldehyde, blocked with goat serum, and incubated overnight at 4°C with antibodies against PPARg polyclonal H-100 (1:100) from Santa Cruz Biotechnology, monoclonal anti-GFAP (1:800), and monoclonal Neu-N (1:500). Sections were incubated with FITC and Texas red secondary fluorescent antibodies and visualized under a confocal laser microscope (Leica TCS NT).
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