RAGE potentiates Aβ-induced perturbation of neuronal function in transgenic mice
2004; Springer Nature; Volume: 23; Issue: 20 Linguagem: Inglês
10.1038/sj.emboj.7600415
ISSN1460-2075
AutoresOttavio Arancio, Huiping Zhang, Xi Chen, Chang Lin, Fabrizio Trinchese, Daniela Puzzo, Shumin Liu, Ashok N. Hegde, Shi Fang Yan, Alan Stern, John Luddy, Lih‐Fen Lue, Douglas G. Walker, Alex E. Roher, Manuel Buttini, Lennart Mucke, Weiying Li, Ann Marie Schmidt, Mark S. Kindy, Paul A. Hyslop, David M. Stern, Shirley Shi Du Yan,
Tópico(s)Nuclear Receptors and Signaling
ResumoArticle30 September 2004free access RAGE potentiates Aβ-induced perturbation of neuronal function in transgenic mice Ottavio Arancio Ottavio Arancio Department of Psychiatry, Physiology and Neuroscience, Dementia Research Center, Nathan Kline Institute, New York University School of Medicine, NY, USA Search for more papers by this author Hui Ping Zhang Hui Ping Zhang Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USAPresent address: Department of Otalaryngology, 1st Affiliated Hospital of Fujian Medical University, Fuzhou, China Search for more papers by this author Xi Chen Xi Chen Department of Neurology, New York University, NY, USA Search for more papers by this author Chang Lin Chang Lin Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Fabrizio Trinchese Fabrizio Trinchese Department of Psychiatry, Physiology and Neuroscience, Dementia Research Center, Nathan Kline Institute, New York University School of Medicine, NY, USA Search for more papers by this author Daniela Puzzo Daniela Puzzo Department of Psychiatry, Physiology and Neuroscience, Dementia Research Center, Nathan Kline Institute, New York University School of Medicine, NY, USA Search for more papers by this author Shumin Liu Shumin Liu Department of Psychiatry, Physiology and Neuroscience, Dementia Research Center, Nathan Kline Institute, New York University School of Medicine, NY, USA Search for more papers by this author Ashok Hegde Ashok Hegde Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC, USA Search for more papers by this author Shi Fang Yan Shi Fang Yan Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Alan Stern Alan Stern Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author John S Luddy John S Luddy Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Lih-Fen Lue Lih-Fen Lue Sun Health Research Institute, Sun City, AZ, USA Search for more papers by this author Douglas G Walker Douglas G Walker Sun Health Research Institute, Sun City, AZ, USA Search for more papers by this author Alex Roher Alex Roher Sun Health Research Institute, Sun City, AZ, USA Search for more papers by this author Manuel Buttini Manuel Buttini Gladstone Institute of Neurological Disease and Department of Neurology, University of California, San Francisco, CA, USA Search for more papers by this author Lennart Mucke Lennart Mucke Gladstone Institute of Neurological Disease and Department of Neurology, University of California, San Francisco, CA, USA Search for more papers by this author Weiying Li Weiying Li Department of Neurosciences, Eli Lilly & Co., Indianapolis, IN, USA Search for more papers by this author Ann Marie Schmidt Ann Marie Schmidt Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Mark Kindy Mark Kindy Department of Physiology and Neuroscience, Neuroscience Institute, Medical University of South Carolina, Charleston, SC, USA Ralph H. Johnson VA Medical Center, Charleston, SC, USA Search for more papers by this author Paul A Hyslop Paul A Hyslop Department of Neurosciences, Eli Lilly & Co., Indianapolis, IN, USA Search for more papers by this author David M Stern David M Stern School of Medicine, Medical College of Georgia, Augusta, GA, USA Search for more papers by this author Shirley Shi Du Yan Corresponding Author Shirley Shi Du Yan Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Ottavio Arancio Ottavio Arancio Department of Psychiatry, Physiology and Neuroscience, Dementia Research Center, Nathan Kline Institute, New York University School of Medicine, NY, USA Search for more papers by this author Hui Ping Zhang Hui Ping Zhang Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USAPresent address: Department of Otalaryngology, 1st Affiliated Hospital of Fujian Medical University, Fuzhou, China Search for more papers by this author Xi Chen Xi Chen Department of Neurology, New York University, NY, USA Search for more papers by this author Chang Lin Chang Lin Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Fabrizio Trinchese Fabrizio Trinchese Department of Psychiatry, Physiology and Neuroscience, Dementia Research Center, Nathan Kline Institute, New York University School of Medicine, NY, USA Search for more papers by this author Daniela Puzzo Daniela Puzzo Department of Psychiatry, Physiology and Neuroscience, Dementia Research Center, Nathan Kline Institute, New York University School of Medicine, NY, USA Search for more papers by this author Shumin Liu Shumin Liu Department of Psychiatry, Physiology and Neuroscience, Dementia Research Center, Nathan Kline Institute, New York University School of Medicine, NY, USA Search for more papers by this author Ashok Hegde Ashok Hegde Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC, USA Search for more papers by this author Shi Fang Yan Shi Fang Yan Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Alan Stern Alan Stern Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author John S Luddy John S Luddy Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Lih-Fen Lue Lih-Fen Lue Sun Health Research Institute, Sun City, AZ, USA Search for more papers by this author Douglas G Walker Douglas G Walker Sun Health Research Institute, Sun City, AZ, USA Search for more papers by this author Alex Roher Alex Roher Sun Health Research Institute, Sun City, AZ, USA Search for more papers by this author Manuel Buttini Manuel Buttini Gladstone Institute of Neurological Disease and Department of Neurology, University of California, San Francisco, CA, USA Search for more papers by this author Lennart Mucke Lennart Mucke Gladstone Institute of Neurological Disease and Department of Neurology, University of California, San Francisco, CA, USA Search for more papers by this author Weiying Li Weiying Li Department of Neurosciences, Eli Lilly & Co., Indianapolis, IN, USA Search for more papers by this author Ann Marie Schmidt Ann Marie Schmidt Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Mark Kindy Mark Kindy Department of Physiology and Neuroscience, Neuroscience Institute, Medical University of South Carolina, Charleston, SC, USA Ralph H. Johnson VA Medical Center, Charleston, SC, USA Search for more papers by this author Paul A Hyslop Paul A Hyslop Department of Neurosciences, Eli Lilly & Co., Indianapolis, IN, USA Search for more papers by this author David M Stern David M Stern School of Medicine, Medical College of Georgia, Augusta, GA, USA Search for more papers by this author Shirley Shi Du Yan Corresponding Author Shirley Shi Du Yan Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA Search for more papers by this author Author Information Ottavio Arancio1, Hui Ping Zhang2, Xi Chen3, Chang Lin2, Fabrizio Trinchese1, Daniela Puzzo1, Shumin Liu1, Ashok Hegde4, Shi Fang Yan2, Alan Stern2, John S Luddy2, Lih-Fen Lue5, Douglas G Walker5, Alex Roher5, Manuel Buttini6, Lennart Mucke6, Weiying Li7, Ann Marie Schmidt2, Mark Kindy8,9, Paul A Hyslop7, David M Stern10 and Shirley Shi Du Yan 2 1Department of Psychiatry, Physiology and Neuroscience, Dementia Research Center, Nathan Kline Institute, New York University School of Medicine, NY, USA 2Departments of Pathology and Surgery, College of Physicians & Surgeons, Columbia University, NY, USA 3Department of Neurology, New York University, NY, USA 4Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC, USA 5Sun Health Research Institute, Sun City, AZ, USA 6Gladstone Institute of Neurological Disease and Department of Neurology, University of California, San Francisco, CA, USA 7Department of Neurosciences, Eli Lilly & Co., Indianapolis, IN, USA 8Department of Physiology and Neuroscience, Neuroscience Institute, Medical University of South Carolina, Charleston, SC, USA 9Ralph H. Johnson VA Medical Center, Charleston, SC, USA 10School of Medicine, Medical College of Georgia, Augusta, GA, USA *Corresponding author. Departments of Pathology and Surgery, Taub Institute for Alzheimer's Disease and the Aging Brain, College of Physicians & Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032, USA. Tel.: +1 212 305 3958; Fax: +1 12 305 5337; E-mail: [email protected] The EMBO Journal (2004)23:4096-4105https://doi.org/10.1038/sj.emboj.7600415 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 (Aβ). 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 Aβ-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 Aβ-induced neuronal perturbation in a model of Alzheimer's-type pathology, and suggest its potential as a therapeutic target to ameliorate cellular dysfunction. Introduction Recent studies have focused attention on amyloid-β peptide (Aβ) as a key pathogenic entity in neuronal dysfunction and neuropathologic changes of Alzheimer's disease (AD). The steady accumulation of Aβ over years in AD may result in progressive displacement of essential neuronal structures. However, early in the disease process, when levels of deposited Aβ are low, mechanisms amplifying and focusing the effects of soluble neurotoxic Aβ assemblies on vulnerable cellular targets could contribute importantly to cellular dysfunction. Several mechanisms could potentially target Aβ to cellular elements. In this regard, cell surface-binding sites are logical to consider for multiple reasons: their capacity to concentrate Aβ at the plasma membrane, where it could directly damage membranes; the possibility that they could function as receptors which engage in intracellular signaling mechanisms; and, their ability to trigger endocytosis, potentially concentrating toxic species in the endolysosomal pathway where disruption of lysosomal integrity could induce severe cellular damage (Yang et al, 1999). As might be expected for a pleiotropic peptide such as Aβ, many cell surface interaction sites have been reported (Snow et al, 1995; Paresce et al, 1996; Yan et al, 1996; Wang et al, 2000). We have focused our attention on Receptor for Advanced Glycation Endproducts (RAGE) for several reasons; its enhanced expression in an Aβ-rich environment (Yan et al, 1995; Lue et al, 2001; Deane et al, 2003); the effectiveness with which it recruits and activates signal transduction mechanisms; and its presence on critical cells (neurons, microglia, and cells of the vessel wall) (Schmidt et al, 2001). A key test of the possible involvement of RAGE (as well as other candidate cell surface binding sites/receptors) in cellular dysfunction associated with Aβ involves analysis of transgenic (Tg) models in which mutant human amyloid precursor protein (mAPP) and/or presenilins are overexpressed (Duff et al, 1996; Hsiao et al, 1996; Holcomb et al, 1998; Mucke et al, 2000). Since many of these Tg models display only subtle changes in neuronal function and neuropathology, especially at early time points, we began our analysis by developing Tg mice with neuronal overexpression of wild-type (wt) RAGE. Our results demonstrate that double Tg mice, with targeted neuronal expression of transgenes for mAPP and RAGE, display functional and pathologic evidence of neuronal perturbation at early times (3–4 months of age), before the steep rise in cerebral Aβ and plaque formation occur. At later times (14–18 months), activation of microglia/astrocytes was significantly increased in double Tgs, compared with single Tg mAPP mice. Consistent with these data, experiments in which Tg mice bearing a dominant-negative form of RAGE (DN-RAGE) targeted to neurons were crossed with Tg mAPP animals (to generate Tg mAPP/DN-RAGE mice) showed preservation of spatial learning/memory and diminished neuropathologic changes compared with Tg mAPP mice. Our data support the possibility that blockade of neuronal RAGE might have cytoprotective effects, especially with respect to preserving neuronal function early in the disease process. Results Generation of Tg RAGE and Tg mAPP/RAGE mice RAGE is a multiligand receptor which binds Aβ in the nanomolar range (apparent Kd's of 50–100 nM; Yan et al, 1996). In vitro, interaction of ligands with RAGE-bearing neuronal-like cell lines induces cell stress, characterized by nuclear translocation of NF-κB, and, eventually, loss of cell viability. In view of increased levels of RAGE in AD brain, including neurons (Yan et al, 1996; Lue et al, 2001), we sought to develop a model system in which neuronal expression of RAGE would be exaggerated so that consequences of receptor–ligand engagement could be assessed in an Aβ-rich environment. A transgene bearing full-length wt human RAGE driven by the platelet-derived growth factor (PDGF) B-chain promoter was constructed and used to generate Tg mice, termed Tg RAGE (or Tg wtRAGE), which were backcrossed into the C57BL/6 strain eight times. Southern blotting from two offspring of the same founder demonstrated the presence of the transgene (Supplementary Figure S1a, lanes 1 and 2). This founder was used to establish a line of Tg animals, which has shown normal gross development, reproductive fitness and lifespan (two other Tg RAGE founders were generated, and one of these was used to develop a second line of Tg RAGE mice). Further characterization of the line shown in panel S1a demonstrated increased expression of RAGE transcripts in total RNA harvested from the cerebral cortex of Tg animals compared with nonTg littermates (Supplementary Figure S1b, lanes 1–4). Western blotting of cortical homogenates confirmed the increase in RAGE expression observed in Tg mice, compared with nonTg littermates (Supplementary Figure S1c, lanes 1 and 2). Immunostaining of neocortex from a Tg mouse demonstrated enhanced expression of RAGE antigen, compared with nonTg littermates (Supplementary Figure S1d). In order to test the effect of neuronal RAGE in an Aβ-rich environment, Tg RAGE mice were crossed with animals expressing a mutant form of human APP (Tg mAPP) also driven by PDGF B-chain promoter, and in the C57BL/6 background (Mucke et al, 2000). The Tg mAPP model was well suited to our strategy of determining whether overexpression of RAGE might enhance/accelerate pathologic changes in the brain, since these animals have been characterized with respect to neuropathologic, biochemical, and physiologic end points (Hsia et al, 1999; Mucke et al, 2000). The cross of Tg RAGE with Tg mAPP mice produced four genotypes, in the expected Mendelian ratios, single Tgs (Tg RAGE, Tg mAPP), double Tgs (Tg mAPP/RAGE), and nonTg littermate controls (Figure 1A). 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). Download figure Download PowerPoint Characterization of transgene expression in Tg mAPP/RAGE mice Expression of RAGE in each of the four genotypes was studied by immunoblotting of homogenates of cerebral cortex with an antibody that recognized both transgene-derived human and endogenous murine RAGE (Figure 1B). Lowest levels of RAGE were observed in nonTg littermates (lane 1), whereas Tg mAPP animals displayed an increase in RAGE antigen (lane 2). This is consistent with enhanced RAGE expression observed in other situations in which the cellular microenvironment is enriched for RAGE ligands, such as AD brain and diabetic vasculature (Lue et al, 2001; Schmidt et al, 2001; Deane et al, 2003). Tg RAGE mice had higher levels of RAGE antigen (Figure 1B1, lane 5) than Tg mAPP animals (Figure 1B1, lane 2), and double Tgs had RAGE levels comparable to Tg RAGE animals (Figure 1B1, lane 4). Levels of RAGE antigen were ∼3-fold greater in Tg mAPP/RAGE mice than in Tg mAPP mice. These results were obtained with animals at 3–4 months of age, and similar differences in RAGE expression were observed at 14–18 months (not shown). Expression of the mAPP transgene was also studied in homogenates of cerebral cortex. Immunoblotting with an antibody selective for human APP revealed comparable expression of the transgene in Tg mAPP and Tg mAPP/RAGE (Figure 1C1, lanes 3–4). As expected, no material immunoreactive with murine monoclonal antibody 6E10 (targeted to human Aβ, residues 1–16) was observed in brain extracts from Tg RAGE or nonTg littermates (Figure 1C1, lanes 1–2). In order to assess the changes in total APP, both endogenous murine and Tg human APP, studies were performed with antibody 369W. Immunoblotting demonstrated similar lower levels of APP in nonTg littermates and Tg RAGE mice (Figure 1C2, lanes 1 and 2), and similar higher levels in Tg mAPP and Tg mAPP/RAGE (Figure 1C, lanes 3 and 4). These results were obtained using animals at 3–4 months of age, and levels of APP remained similar up to the oldest age group analyzed (14–18 months). Cellular stress in Tg mAPP/RAGE mice: activation of NF-κB and inflammation NF-κB activation has been identified in AD brain (Yan et al, 1996; Kaltschmidt et al, 1997). Based on the hypothesis that a receptor for Aβ should magnify cell stress at early stages of Aβ accumulation, nuclear binding activity for NF-κB was studied by gel shift analysis using nuclear extracts from cerebral cortex with a consensus 32P-labeled NF-κB probe. At 3–4 months of age, Tg mAPP/RAGE mice displayed a strong gel shift band compared with a virtually undetectable signal in nonTg littermates and single Tgs (Figure 2A1). The data shown in Figure 2A2 are representative of results obtained in mice and densitometry of autoradiograms from this larger group confirmed the striking increase in nuclear translocation of NF-κB in double Tgs. 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). Download figure Download PowerPoint In view of the association of NF-κB activation with inflammation, we considered the possibility that neurons in double Tg mice might incite an inflammatory response, even as early as 3–4 months. Thus, we compared microgliosis and astrocytosis in Tg mAPP/RAGE and Tg mAPP animals. Microglia were visualized immunohistochemically with antibodies to CD11b (complement receptor 3) or phosphotyrosine, and astrocytes were visualized with antibody to glial fibrillary acidic protein (GFAP). The results of image analysis of these sections are shown in the histogram in Figure 2, panel B, and representative micrographs are shown in panels c–f (animals were 14–18 months). Analysis of Tg mAPP/RAGE mice at 3–4 and 7–8 months of age did not reveal significantly increased microgliosis or astrocytosis compared with Tg mAPP, Tg RAGE or nonTg littermates (not shown). However, at 14–18 months, Tg mAPP/RAGE mice had significantly more plaque-associated reactive microglia and astrocytes (P⩽0.015; Figure 2B). It is possible that the latter reflect an exaggerated secondary inflammatory response to deposited Aβ. Perturbation of neuronal function in Tg mAPP/RAGE mice In view of accentuated cell stress in brains of Tg mAPP/RAGE mice, as reflected by NF-κB activation at early times, we sought to determine whether this would be reflected in impaired spatial learning/memory in the radial arm water maze (Diamond et al, 1999). At 3–4 months of age, nonTg littermates showed strong learning and memory capacity (Figure 3A). Similar results were obtained with Tg RAGE, as well as Tg mAPP mice (Figure 3A). In contrast, Tg mAPP/RAGE mice averaged about four errors by trials 4 or 5, indicative of impaired spatial memory for platform location between trials, as well as during the 30 min delay before trial 5 (Figure 3A). To determine whether combined overexpression of RAGE and mAPP impaired vision, motor coordination or motivation, we also tested mice with a visible platform task. The four genotypes showed no difference in their speed of swimming (Supplementary Figure S2a1) or in the time required to reach the platform in this version of the task (Supplementary Figure S2a2). At 5–6 months, the deficit in learning/memory in Tg mAPP/RAGE mice was worse (≈8 errors by trial 4 or 5) than in younger double Tg animals (Figure 3B). A deficit, though less severe, was also observed in age-matched Tg mAPP mice (≈6 errors by trials 4 or 5), whereas single Tg RAGE mice and nonTg littermates showed intact spatial memory (Figure 3B). Spatial memory impairment persisted in older double Tg animals (12–13 months; Supplementary Figure S3); their performance was worse than single Tg mAPP mice, Tg RAGE mice, and nonTg littermates. 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). (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). Download figure Download PowerPoint These results indicate an early perturbation of learning and memory, consequent to overexpression of RAGE and mAPP. Since cognitive abnormalities in AD are thought to be linked to synaptic dysfunction (Selkoe, 2002), we examined synaptic transmission under basal conditions and during long-term potentiation (LTP). Consistent with previous observations (Hsia et al, 1999), field-excitatory post-synaptic potential (fEPSPs) in the CA1 stratum radiatum revealed impaired basal synaptic transmission (BST) in young (3–5 months) double Tg mice (≈24% decrease in slope of the input–output curve) and Tg mAPP littermates (≈26% decrease) (Figure 4A). BST was further reduced in aged (8–10 months) Tg mAPP/RAGE (64% decrease) and Tg mAPP animals (≈58% decrease) (Figure 4A). Both younger and older Tg mice overexpressing RAGE alone did not show any significant difference in BST, compared with nonTg littermates (Figure 4A). CA1 LTP was normal in young (3–5 months) Tg mice (all genotypes) and nonTg littermates (Figure 4B). However, LTP was most severely affected in 8–10-month-old double Tg mice compared with the other groups (Figure 4C). At 60 min after LTP induction, we observed a 140±9% increase in potentiation in Tg mAPP/RAGE mice, whereas nonTg littermates displayed a 202±13% increase. Single Tg RAGE and Tg mAPP showed levels of LTP comparable to nonTg animals (188±20 and 203±17%, respectively). These findings are consistent with the hypothesis that RAGE-Aβ interaction alters synaptic plasticity. In addition, since BST is affected both in single Tg mAPP and double Tg mice, but LTP is reduced only in the double Tgs, we infer that overexpression of RAGE in an Aβ-rich environment further perturbs synaptic function in the older mice, impairing plasticity. 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 m
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