O4-06-07: Involvement of the prostaglandin E2 receptors, EP2 and EP4, in the production of amyloid-β peptides both in vitro and in vivo

T198 O4-06-07

Plenary PL-05 INVOLVEMENT OF THE PROSTAGLANDIN E2 RECEPTORS, EP2 AND EP4, IN THE PRODUCTION OF AMYLOID-␤ PEPTIDES BOTH IN VITRO AND IN VIVO

Tatsuya Hoshino1, Tadashi Nakaya2, Yukihiko Sugimoto3, Wataru Araki4, Shuh Narumiya3, Toshiharu Suzuki2, Tohru Mizushima1, 1 Kumamoto University, Kumamoto, Japan; 2Hokkaido University, Sapporo, Japan; 3Kyoto University, Kyoto, Japan; 4National Institute of Neuroscience, Kodaira, Japan. Contact e-mail: [email protected] Background: Amyloid-␤ peptides (A␤, generated by proteolysis of the ␤-amyloid precursor protein (APP) by ␤- and ␥-secretases, play an important role in the pathogenesis of Alzheimer’s disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Methods: Here we show that prostaglandin E2 (PGE2), a strong inducer of inflammation, stimulates the production of A␤ in cultured human cells, which express a mutant type of APP (APPsw). We have demonstrated using subtypespecific agonists that, of the four main subtypes of PGE2 receptors (EP1-4). Results: EP2 and EP4 receptors are responsible for this PGE2-stimulated production of A␤. An EP4 receptor antagonist suppressed the PGE2stimulated production of A␤ in cells lacking the function of EP2. Activation of EP2 and EP4 receptors is coupled with an increase in cellular cAMP levels and activation of protein kinase A (PKA), and we found that an inhibitor for adenylate cyclase or PKA suppresses the EP2-mediated but not EP4-mediated stimulation of the production of A␤ by PGE2. On the other hand, either sucrose or siRNA for clathrin, which inhibits the endocytosis, suppresses the EP4-mediated but not EP2-mediated stimulation of the production of A␤ by PGE2. By immunostaining, we observed PGE2dependent internalization of EP4 receptor and cells expressing mutant EP4 receptor without the internalization activity was inert for PGE2-stimulated production of A␤. Immunoprecipitation assay revealed the physical interaction between EP4 receptor and presenillin-1 and internalization of presenillin-1 was observed depending both PGE2 and EP4 receptor. Experiments of labeling of membrane protein by biotin also supported the notion that presenillin-1 is internalized into cells via endosome, accompanying with EP4 receptor. Transgenic mice expressing APPsw showed lower levels of A␤ in the brain, when they were crossed with mice lacking either EP2 or EP4 receptors. Conclusions: All these results suggest that both EP4 receptor-dependent internalization of presenillin-1 (and ␥-secretase complex) and EP2 receptor-dependent activation of adenylate cyclase and PKA involve PGE2-stimulated production of A␤ in vitro, and PGE2-mediated activation of EP2 and EP4 receptors is involved in the production of A␤ in vivo and in the pathogenesis of AD. O4-06-08

IDENTIFICATION AND CHARACTERIZATION OF A NOVEL APP MUTATION (E693⌬) IN FAMILIAL ALZHEIMER’S DISEASE

Takami Tomiyama1, Tetsu Nagata2, Hiroshi Takuma1, Hiroyuki Shimada1, Rie Teraoka1, Akiko Fukushima1, Hyoue Kanemitsu1, Ryozo Kuwano3, Masaki Imagawa4, Suzuka Ataka1,5, Yasuhiro Wada1,5, Eito Yoshioka1,5, Tomoyuki Nishizaki2, Yasuyoshi Watanabe1,5, Hiroshi Mori1, 1Osaka City University Graduate School of Medicine, Osaka, Japan; 2Hyogo College of Medicine, Nishinomiya, Japan; 3Niigata University, Niigata, Japan; 4Imagawa Clinic, Osaka, Japan; 5RIKEN, Kobe, Japan. Contact e-mail: [email protected] Background: Based on experimental models, soluble A␤ oligomers have been proposed to initiate synaptic and cognitive dysfunction in Alzheimer’s disease. However, there is no direct evidence in humans that this mechanism can cause Alzheimer’s disease. We identified the new type of APP mutation that lacks codon 693 (GAA) encoding glutamate-22 in the A␤ sequence in several Japanese pedigrees showing AD. Methods: We studied the effects of this mutation on A␤ production, degradation, aggregation, and toxicity. Production of A␤ was determined by the amounts of

A␤ secretion from cells transfected with APP constructs. Degradation, aggregation, and toxicity of A␤ were examined using synthetic A␤ peptides. Results: While the secretion of total A␤ from cells was markedly reduced by this mutation, the mutant A␤ E22⌬ peptide was more resistant to degradation by neprilysin and insulin-degrading enzyme. The A␤ E22⌬ peptide showed a unique aggregation property of enhanced oligomerization but no fibrillization. When injected into rat cerebral ventricle (10 ng peptide/2 ␮l/rat), the A␤1-42 E22⌬ peptide inhibited hippocampal longterm potentiation more potently than wild-type A␤1-42 peptide in vivo. In the MTT assay, wild-type A␤1-42 peptide showed a dose-dependent (1 nM to 1 ␮M) cytotoxicity to cultured neuronal cells, whereas A␤1-42 E22⌬ peptide was toxic only at 1 ␮M. In mouse hippocampal slices, however, A␤1-42 E22⌬ peptide caused a dose-dependent (0.1-10 ␮M) decrease of synaptophysin, whereas wild-type A␤1-42 peptide was trophic at 0.1-1 ␮M and toxic at 10 ␮M. Thus, extracellular A␤ E22⌬ was shown to induce more potent synaptic dysfunction and alteration, but lower neurodegeneration, than wild-type A␤ probably due to its unique aggregation property. Consistent with the non-fibrillogenic property of the mutant A␤, a very low amyloid signal was observed in the patient’s brain on positron emission tomography using Pittsburgh compound-B (see also Shimada et al. in this meeting). Conclusions: These results suggest that the E693⌬ mutation causes disease by enhanced formation of synaptotoxic A␤ oligomers in the absence of fibril formation. Our findings may provide genetic validation in humans for the A␤ oligomer hypothesis. THURSDAY, JULY 31, 2008 PLENARY PL-05 PL-05-01

BRAIN RESERVE: THE EPIDEMIOLOGICAL PERSPECTIVE

David A. Bennett, Rush Alzheimer’s Disease Center, Chicago, IL, USA. Contact e-mail: [email protected] Background: It has long been recognized that some type of reserve can protect the nervous system from expressing AD pathology as cognitive impairment and dementia. However, little data are available from humans regarding the neurobiologic basis of reserve and the factors that determine the ability of the nervous system to tolerate AD pathology. Methods: Data are presented from two, large, longitudinal epidemiologic clinical-pathologic studies, the Religious Orders Study and the Rush Memory and Aging Project, designed to identify risk factors for incident AD and investigate biologic pathways linking risk factors to clinical disease. Incident AD is diagnosed by NINCDS/ADRDA criteria and 19 cognitive function tests are used to summarize global cognition and five domains (episodic memory, semantic memory, working memory, perceptual speed, and visuospatial ability). AD pathology was quantified by counts of neuritic plaques, diffuse plaques, and neurfibrillary tangles based on silve stain, amyloid load by image analysis, and tangle density by stereology. The number, age, volume and location of macrospic cerebral infarcts were recored. The number and location of alpha-synuclein positive Lewy bodies were recorded. Results: Data from these studies suggest the existence of three types of brain reserve. First, evidence is presented demonstrating that common co-existing neuropathology (e.g., cerebral infarcts and Lewy bodies) reduce brain reserve capacity and that some risk factors for clinical AD (e.g., diabetes) are related to coexisting pathology rather than to AD pathology. Second, some risk factors for incident AD (e.g., social networks) are not directly related to measures of neuropathology but modify the relation of neuropathology to clinical AD, consistent with neural efficiency or compensation. Finally, some risk factors for incident AD (e.g., psychological distress) are unrelated to measures of neuropathology and work through as yet completely unknown mechanisms to reduce brain reserve. Conclusions: These data suggest a variety of approaches to the prevention of AD including reducing the occurrence of coexisting pathology, identifying factors that allow one to tolerate AD pathology, and using these risk factor clinical disease associations to identify novel targets for therapy.