RAGE potentiates Abeta-induced perturbation of neuronal function in transgenic mice - PubMed (original) (raw)

Comparative Study

. 2004 Oct 13;23(20):4096-105.

doi: 10.1038/sj.emboj.7600415. Epub 2004 Sep 30.

Hui Ping Zhang, Xi Chen, Chang Lin, Fabrizio Trinchese, Daniela Puzzo, Shumin Liu, Ashok Hegde, Shi Fang Yan, Alan Stern, John S Luddy, Lih-Fen Lue, Douglas G Walker, Alex Roher, Manuel Buttini, Lennart Mucke, Weiying Li, Ann Marie Schmidt, Mark Kindy, Paul A Hyslop, David M Stern, Shirley Shi Du Yan

Affiliations

Comparative Study

RAGE potentiates Abeta-induced perturbation of neuronal function in transgenic mice

Ottavio Arancio et al. EMBO J. 2004.

Abstract

Receptor for Advanced Glycation Endproducts (RAGE), a multiligand receptor in the immunoglobulin superfamily, functions as a signal-transducing cell surface acceptor for amyloid-beta peptide (Abeta). In view of increased neuronal expression of RAGE in Alzheimer's disease, a murine model was developed to assess the impact of RAGE in an Abeta-rich environment, employing transgenics (Tgs) with targeted neuronal overexpression of RAGE and mutant amyloid precursor protein (APP). Double Tgs (mutant APP (mAPP)/RAGE) displayed early abnormalities in spatial learning/memory, accompanied by altered activation of markers of synaptic plasticity and exaggerated neuropathologic findings, before such changes were found in mAPP mice. In contrast, Tg mice bearing a dominant-negative RAGE construct targeted to neurons crossed with mAPP animals displayed preservation of spatial learning/memory and diminished neuropathologic changes. These data indicate that RAGE is a cofactor for Abeta-induced neuronal perturbation in a model of Alzheimer's-type pathology, and suggest its potential as a therapeutic target to ameliorate cellular dysfunction.

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Figures

Figure 1

Figure 1

Identification and characterization of Tg mAPP/RAGE mice. (A) PCR analysis of tail DNA from one litter (+, RAGE or mAPP transgene present; −, transgene absent). (B) Western analysis of RAGE expression in the cerebral cortex of the four genotypes of mice. Homogenates of cerebral cortex (100 μg) were subjected to SDS–PAGE (reduced; 10%), followed by immunoblotting with anti-RAGE IgG (6.5 μg/ml) (B1). Densitometric analysis of gels is shown in (B2) (nonTg, _n_=7; Tg RAGE, _n_=5; Tg mAPP, _n_=7; Tg mAPP/RAGE, _n_=4). Results are means±s.e. (C) Western analysis of APP expression in the cerebral cortex of the four genotypes of mice. Homogenates of cortical protein were prepared as above and subjected to SDS–PAGE (reduced; 7.5%), followed by immunoblotting with either antibody 6E10 (C1; 1 μg/ml) or 369W (C2; 1:2000 dilution).

Figure 2

Figure 2

Activation of NF-κB and inflammation in Tg mAPP/RAGE mice. (A) Nuclear translocation of NF-κB was assessed in nuclear extracts from cerebral cortex (10 μg total protein/lane) of mice (age 3–4 months) with 32P-labeled consensus NF-κB probe (A1). The adjacent panel displays image analysis of results from a larger group of mice (4–6 mice/genotype) (A2). Panel A3 displays results of NF-κB gel shift experiments with Tg mAPP/RAGE, Tg mAPP/DN-RAGE, and nonTg littermates. Lane 1 shows migration of free probe. (B) Microgliosis and astrocytosis in brains of Tg mAPP/RAGE mice compared with the other groups. Results of image analysis are shown at 14–18 months in the cerebral cortex and hippocampus (4–6 mice/genotype). (C–F) Representative sections from Tg mAPP/RAGE (C, D) and Tg mAPP (E, F) mice at age 14–18 months to demonstrate microglia (C, E) and astrocytes (D, F). The arrows denote cells reactive with the indicated markers. Scale bar=10 μm. Multiple images similar to those in (C–F) were used to construct the histogram shown in panel (B).

Figure 3

Figure 3

Functional neuronal deficits: spatial learning and memory in Tg mAPP/RAGE and Tg mAPP/DN-RAGE mice. (A, B) Spatial learning and memory was tested in the radial arm water maze at 3–4 (A) and 5–6 months of age (B) in mice of the indicated genotypes (in (A), _n_=7–8 male mice/genotype; in (B), _n_=5 male mice/genotype): Tg mAPP/RAGE (APP/RAGE), nonTg littermate (nonTg), Tg mAPP (APP), and Tg RAGE (RAGE). (A1–A4) denote the acquisition trials, and R denotes the retention trial. In panel A, **P<0.01 Tg mAPP/RAGE compared with nonTg mice (by repeated-measure ANOVA followed by Fisher's protected least significant difference for _post hoc_ comparisons in this and the following graphs). In panel B, **_P_<0.01 Tg mAPP/RAGE and Tg mAPP mice compared with nonTg mice; #_P_<0.01 Tg mAPP/RAGE compared with Tg mAPP mice. ANOVA revealed a main age effect in Tg mAPP/RAGE mice and Tg mAPP mice (_P_<0.05 for both), but not in nonTg and DN-RAGE mice (_P_>0.05 for both). (C, D) Effect of DN-RAGE transgene on spatial learning and memory in Tg mice at 3–4 months (_n_=5–8 male mice/genotype (C)) and 5–6 months (_n_=6–9 male mice/genotype (D)). The following genotypes were tested: nonTg littermate (nonTg), Tg DN-RAGE (DN-RAGE), Tg mAPP (APP), and Tg mAPP/DN-RAGE (APP/DN-RAGE). In panel D, **P<0.01 Tg mAPP mice compared with nonTg mice; #_P_<0.01 Tg mAPP mice compared with Tg mAPP/DN-RAGE mice, and *_P_<0.05 Tg mAPP/DN-RAGE mice compared with nonTg littermates. ANOVA revealed a main age effect in Tg mAPP mice (_P_<0.05) but not in Tg mAPP/DN-RAGE, DN-RAGE, and nonTg mice (_P_>0.05).

Figure 4

Figure 4

Functional neuronal deficits: decreased LTP in Tg mAPP/RAGE mice. (A) BST was reduced in the CA1 region of slices from 3–5-month-old Tg mAPP/RAGE (APP/RAGE) mice (*P<0.05) and Tg mAPP (APP) littermates compared with nonTg littermates (male animals were used throughout in electrophysiologic studies). A greater reduction in BST was observed in 10-month-old Tg mAPP/RAGE mice and Tg mAPP littermates (**P<0.01). Single Tg RAGE (RAGE) mice showed normal values of BST at both ages. The responsiveness of CA1 cells to increasing afferent fiber stimulation (slope of input–output (i/o) relation) was measured to assess BST strength. Results from 3–5-month-old animals were based on recordings in 16 slices from five male Tg mAPP/RAGE mice, 19 slices from six Tg mAPP mice, 16 slices from six Tg RAGE mice, and 19 slices from seven nonTg mice. Results from 8–10-month-old mice were based on recordings in 12 slices from five Tg mAPP/RAGE mice; 10 slices from four Tg mAPP mice; 15 slices from six Tg RAGE; and 12 slices from six nonTg mice. Insets show representative fEPSP for a nonTg mouse and a Tg mAPP/RAGE mouse, illustrating that far higher stimulation strengths are required to elicit synaptic responses in 10-month-old Tg mAPP/RAGE mice. Scale: 0.2 mV; 5 ms. (B) LTP was normal in the CA1 region of slices from 3–5-month-old Tg mice (all genotypes) and their nonTg littermates. Theta-burst stimulation indicated by the three arrows was delivered after recording a 15-min baseline (_n_=16 slices from five Tg mAPP/RAGE mice, _n_=19 slices from six male Tg mAPP mice, _n_=16 slices from six male Tg RAGE mice, and _n_=19 slices from seven male nonTg mice). Insets show traces taken 1 min before and 60 min after LTP induction on 3–5-month-old nonTg and Tg mAPP/RAGE mice. Scale: 0.1 mV (nonTg), 0.05 mV (Tg mAPP/RAGE mice); 2.5 ms. (C) LTP was reduced in the CA1 region of slices from 8–10-month-old double Tg mAPP/RAGE mice, whereas Tg mAPP, Tg RAGE, and nonTg mice showed normal potentiation (_n_=12 slices from five Tg mAPP/RAGE mice; _n_=10 slices from four Tg mAPP mice; _n_=15 slices from six Tg RAGE mice; and _n_=12 slices from six nonTg mice; *P<0.05). Insets show traces taken 1 min before and 60 min after LTP induction on 8–10-month-old nonTg and Tg mAPP/RAGE mice. Scale: 0.1 mV (nonTg), 0.03 mV (Tg mAPP/RAGE mice); 2.5 ms. (D) LTP was still reduced in double Tg mAPP/RAGE mice after enhancement of the theta-burst strength (_n_=7 slices from four Tg mAPP/RAGE mice; and _n_=8 slices from four nonTg mice; *P<0.05).

Figure 5

Figure 5

Characterization of Tg mAPP/RAGE and Tg mAPP/DN-RAGE mice: effect on AChE activity. (A1, A2) AChE activity was determined histochemically in the subiculum of mice of the indicated genotype at 3–4 (A1; _n_=4–9/group) and 14–18 months (A2; _n_=5–12/group) of age: nonTg littermate (nonTg), Tg RAGE (RAGE), Tg mAPP (mAPP), and Tg mAPP/RAGE (mAPP/RAGE). The bar graph shows the results of image analysis. (B) Representative images from AChE histochemical staining from the subiculum of mice of the indicated genotype from the experiment in A1. Scale bar=10 μm. (C1, C2) AChE activity was determined as above in the indicated mice (nonTg, Tg DN-RAGE (DN-RAGE), Tg mAPP (mAPP), and Tg mAPP/DN-RAGE (mAPP/DN-RAGE)) at 3–4 months (C1; _n_=7–12/group) and 14–18 months of age (C2; _n_=7–12/group). (D) Representative images of AChE staining for the indicated mice from the experiment in C2. Scale bar=10 μm. (E) Area occupied by synaptophysin immunoreactivity in the stratum lacunosum moleculare of the hippocampus in mice of the indicated genotype at 3–4 months of age (mean±s.e. are shown).

Figure 6

Figure 6

Phosphorylation/activation of CREB (A), MAP kinases (B, C), and CAMKII (D) in Tg mice. (A) Protein extracts were obtained from hippocampii of mice of the indicated genotypes (Tg mAPP, Tg (wt)RAGE, Tg mAPP/RAGE, Tg mAPP/DN-RAGE, nonTg) at 3–4 months of age and subjected to immunoblotting with antibody to total CREB or phospho (p)-CREB. The upper panel displays quantitation of the results with phospho-CREB by image analysis (_n_=5–8/group) and the lower panel shows representative immunoblots. (B, C) The experiment was performed as in (A), except that antibodies for immunoblotting were anti-total p38 and anti-phospho-p38 IgG (B), and anti-total Erk1/2 and anti-phospho-Erk1/2 IgG (C). The upper panel displays image analysis of results with the phospho antibodies and _n_=5–8/group (in the case of Erk, image analysis was performed on p44). In panel D, the experiment was performed with antibodies to total and phospho-CAMKII (CAMKII) and the animals were 14–18 months of age (image analysis shows results of phospho-CAMKII from 5–13 animals/group).

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