- Email: [email protected]
ALTERED LIPID MEDIATORS AND RECEPTORS OF RESOLUTION IN THE ENTORHINAL CORTEX OF ALZHEIMER'S DISEASE
P338
Poster Presentations: P1
Alzheimer’ disease pathogenesis. Primary murine microglia that lack prostaglandin E 2 receptor subtype 2 (EP2) have a dual phenotype of decreased innate immune-mediated neurotoxicity and increased Ab peptide phagocytosis, features that have been replicated in vivo. Here we tested the hypothesis that expression and activity of scavenger receptor CD36 influences EP2-regulated A b phagocytosis. Methods: Primary microglia were isolated from brains of newborn C57BL/6 mice. CD36 mRNA and protein levels were assessed by quantitative real-time PCR and flow cytometry, respectively.Ab 42 phagocytosis by primary microglia was determined by incubation with pHrodo-labeled Ab 42 followed by flow cytometry. CD36 mRNA levels were measured in hippocampus of mice following intracerebroventricular (ICV) injection of PIC, TLR3 activator. Results: Treatment of microglia with the EP2 agonist Butaprost reduced CD36 expression, as measured by mRNA and cell surface protein levels, and inhibited microglial A b 42 phagocytosis. Consistent with these findings, preventing EP2 activation by both pharmacological and genetic approaches increased microglial CD36 expression as well as A b 42 phagocytosis. Activation of the innate immune response by TLR activators, such as TLR3, TLR4 and TLR7, similarly reduced CD36 expression and phagocytic capacity in primary microglia. These effects were reduced by at least half by an EP2 antagonist, AH 6809, with the microglia Ab 42 phagocytosis effect completely dependent on CD36 activity. Finally, our in vivo studies demonstrated that hippocampal CD36 mRNA levels were significantly reduced following ICV injection of theTLR3 activator PIC; this effect was reversed by an EP2 antagonist. Conclusions: Our results are consistent with those of other studies in supporting a key role for CD36 in Ab phagocytosis by microglia and highlight selective EP2 antagonists as an effective means to suppress this toxic reinforcing cycle. Our findings suggest that EP2 antagonists have potential as a therapeutic approach to suppressing key aspects of AD pathogenesis. P1-099
ALTERED LIPID MEDIATORS AND RECEPTORS OF RESOLUTION IN THE ENTORHINAL CORTEX OF ALZHEIMER’S DISEASE
Xiuzhe Wang1, Ann-Charlotte Granholm2, Jonathan M. Fitzgerald3, Charles N. Serhan3, Marianne Schultzberg1, 1Karolinska Institutet, Stockholm, Sweden; 2Medical University of South Carolina, Charleston, South Carolina, United States; 3Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, United States. Contact e-mail: [email protected] Background: The role of inflammation in Alzheimer’s disease (AD) is not fully elucidated. Resolution of inflammation and its lipid mediators have emerged as potential targets in diseases associated with inflammation. We have recently demonstrated reduced levels of specialized pro-resolving mediators (SPMs) in AD hippocampus and cerebrospinal fluid. In the present study, we investigated SPMs, other lipid mediators, and receptors involved in resolution, in the entorhinal cortex from AD and control subjects. Methods: Postmortem samples of entorhinal cortex were collected from 6 AD and 5 non-AD brains for analysis of lipid mediators. The samples were extracted and analyzed by liquid chromatography - tandem mass spectrometry (LC-MS-MS). SPM receptors were analyzed by immunohistochemistry in 5 AD and 6 non-AD cases. Results: The levels of SPMs including maresin 1 (MaR1), protectin D1 (PD1) and lipoxin B4 (LXB 4) were lower in the AD group than in controls. In contrast, resolvin E1 (RvE1) levels were higher in AD than in controls. The levels of proinflammatory molecules such as prostaglandin E2 (PGE 2) and thromboxane B2 (TxB 2) were higher in AD. Levels of 17-hydroxy-docosahexaenoic acid (17-HDHA), which is a biosynthetic pathway marker of MaR1, PD1 and resolvin Ds (RvDs), were decreased in AD, while another DHA metabolite, 4-HDHA, was increased in AD. Immunohistochemical staining of LXA 4 receptor/formyl peptide receptor 2 (ALX/FPR2), and chem-
erin receptor 23 (ChemR23, the receptor for RvE1), was predominant in glia and neurons in layer I-III of the entorhinal cortex. The glial and neuronal staining in AD was different compared to controls, and the difference varied between the different layers. Conclusions: These data demonstrated that lipid mediators involved in resolution were dysregulated in entorhinal cortex of the AD brain. Decreased levels of SPMs, including maresin, protectin and lipoxin series in the AD entorhinal cortex, are consistent with our previous findings in the hippocampus. However, one of the SPMs, RvE1, for the first time shown to occur in brain, was increased in AD. Further characterization is required for fully understanding the function of different SPMs in healthy brain and in AD.
P1-100
PREVENTIVE EFFECTS OF DAIRY PRODUCTS FERMENTED WITH PENICILLIUM CANDIDUM ON ALZHEIMER’S DISEASE AND IDENTIFICATION OF A NOVEL OLEAMIDE WITH ENHANCED MICROGLIAL PHAGOCYTOSIS AND ANTI-INFLAMMATORY ACTIVITY
Yasuhisa Ano1, Makiko Ozawa2, Toshiko Kutsukake1, Shinya Sugiyama3, Kazuyuki Uchida2, Aruto Yoshida1, Hiroyuki Nakayama2, 1Kirin Company, Ltd., Kanagawa, Japan; 2The University of Tokyo, Tokyo, Japan; 3Koiwai Dairy Products Co., Ltd., Iwate, Japan. Contact e-mail: Yasuhisa_Ano@ kirin.co.jp Background: Despite the ever-increasing number of dementia patients worldwide, fundamental therapeutic approaches have not been established yet. Preventive measures to the disease in daily life including diet, nutrition, exercise, and learning activity continue to raise attention. Several epidemiological studies suggest that intake of fermented dairy products prevents cognitive decline in the elderly. However, the mechanism and the active compounds remain to be investigated. In this study we evaluated the preventive effects of fermented dairy products on dementia using Alzheimer’s disease model (5xFAD) animals with chronic inflammation and identified active compounds in dairy products. Methods: Camembert cheeses made by conventional fermentation with P. candidum weredelipidated and used as samples for diet preparation. Three-month old 5xFAD mice were fed diet with or without the samples (2% w/w) for three months. The mice were then sacrificed, and their b amyloid (Ab) accumulations, neurotrophic factors and cytokine productions were measured by ELISA and immunohistochemical methods. To identify the active component, primary microglia isolated from newborn murine brains were pretreated with fractionated sample extractions, and then treated with lipopolysaccharide and IFN-g for evaluation of cytokine productions and cell surface marker expressions by ELISA and flow cytometry, or treated with Ab-FAM for phagocytosis assay. Results: From among the cheeses examined, Camembert types fermented with P. candidum showed strikingly high anti-inflammatory activity. In the parallel-group study using 5xFAD mice, Ab accumulation and TNF-a and MIP-1a productions in the group fed the sample diet were significantly restrained. Production of hippocampal BDNF and GDNF and synaptophysin content increased in 5xFAD mice fed the samples. In addition, oleamide was identified as an active compound enhancing both micoglial phagocytosis and anti-inflammatory activity. Enhanced phagocytosis by oleamide was diminisched by PPAR-g antagonist treatment. Oleamide also induced M2 type CD206+CD11b+microglia and suppressed the production of TNF-a-and MIP-1a, and the expression of CD68, CD80 and CD86. Conclusions: The present study demonstrated the epidemiologically reported preventive effects of dairy products on Alzheimer’s disease. Oleamide identified as an active content in the P. candidum fermented dairy products reduces Ab accumulation and suppresses the induced chronic inflammation following Ab deposits, which might be helpful for the prevention of dementia.
Poster Presentations: P1
Alzheimer’ disease pathogenesis. Primary murine microglia that lack prostaglandin E 2 receptor subtype 2 (EP2) have a dual phenotype of decreased innate immune-mediated neurotoxicity and increased Ab peptide phagocytosis, features that have been replicated in vivo. Here we tested the hypothesis that expression and activity of scavenger receptor CD36 influences EP2-regulated A b phagocytosis. Methods: Primary microglia were isolated from brains of newborn C57BL/6 mice. CD36 mRNA and protein levels were assessed by quantitative real-time PCR and flow cytometry, respectively.Ab 42 phagocytosis by primary microglia was determined by incubation with pHrodo-labeled Ab 42 followed by flow cytometry. CD36 mRNA levels were measured in hippocampus of mice following intracerebroventricular (ICV) injection of PIC, TLR3 activator. Results: Treatment of microglia with the EP2 agonist Butaprost reduced CD36 expression, as measured by mRNA and cell surface protein levels, and inhibited microglial A b 42 phagocytosis. Consistent with these findings, preventing EP2 activation by both pharmacological and genetic approaches increased microglial CD36 expression as well as A b 42 phagocytosis. Activation of the innate immune response by TLR activators, such as TLR3, TLR4 and TLR7, similarly reduced CD36 expression and phagocytic capacity in primary microglia. These effects were reduced by at least half by an EP2 antagonist, AH 6809, with the microglia Ab 42 phagocytosis effect completely dependent on CD36 activity. Finally, our in vivo studies demonstrated that hippocampal CD36 mRNA levels were significantly reduced following ICV injection of theTLR3 activator PIC; this effect was reversed by an EP2 antagonist. Conclusions: Our results are consistent with those of other studies in supporting a key role for CD36 in Ab phagocytosis by microglia and highlight selective EP2 antagonists as an effective means to suppress this toxic reinforcing cycle. Our findings suggest that EP2 antagonists have potential as a therapeutic approach to suppressing key aspects of AD pathogenesis. P1-099
ALTERED LIPID MEDIATORS AND RECEPTORS OF RESOLUTION IN THE ENTORHINAL CORTEX OF ALZHEIMER’S DISEASE
Xiuzhe Wang1, Ann-Charlotte Granholm2, Jonathan M. Fitzgerald3, Charles N. Serhan3, Marianne Schultzberg1, 1Karolinska Institutet, Stockholm, Sweden; 2Medical University of South Carolina, Charleston, South Carolina, United States; 3Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, United States. Contact e-mail: [email protected] Background: The role of inflammation in Alzheimer’s disease (AD) is not fully elucidated. Resolution of inflammation and its lipid mediators have emerged as potential targets in diseases associated with inflammation. We have recently demonstrated reduced levels of specialized pro-resolving mediators (SPMs) in AD hippocampus and cerebrospinal fluid. In the present study, we investigated SPMs, other lipid mediators, and receptors involved in resolution, in the entorhinal cortex from AD and control subjects. Methods: Postmortem samples of entorhinal cortex were collected from 6 AD and 5 non-AD brains for analysis of lipid mediators. The samples were extracted and analyzed by liquid chromatography - tandem mass spectrometry (LC-MS-MS). SPM receptors were analyzed by immunohistochemistry in 5 AD and 6 non-AD cases. Results: The levels of SPMs including maresin 1 (MaR1), protectin D1 (PD1) and lipoxin B4 (LXB 4) were lower in the AD group than in controls. In contrast, resolvin E1 (RvE1) levels were higher in AD than in controls. The levels of proinflammatory molecules such as prostaglandin E2 (PGE 2) and thromboxane B2 (TxB 2) were higher in AD. Levels of 17-hydroxy-docosahexaenoic acid (17-HDHA), which is a biosynthetic pathway marker of MaR1, PD1 and resolvin Ds (RvDs), were decreased in AD, while another DHA metabolite, 4-HDHA, was increased in AD. Immunohistochemical staining of LXA 4 receptor/formyl peptide receptor 2 (ALX/FPR2), and chem-
erin receptor 23 (ChemR23, the receptor for RvE1), was predominant in glia and neurons in layer I-III of the entorhinal cortex. The glial and neuronal staining in AD was different compared to controls, and the difference varied between the different layers. Conclusions: These data demonstrated that lipid mediators involved in resolution were dysregulated in entorhinal cortex of the AD brain. Decreased levels of SPMs, including maresin, protectin and lipoxin series in the AD entorhinal cortex, are consistent with our previous findings in the hippocampus. However, one of the SPMs, RvE1, for the first time shown to occur in brain, was increased in AD. Further characterization is required for fully understanding the function of different SPMs in healthy brain and in AD.
P1-100
PREVENTIVE EFFECTS OF DAIRY PRODUCTS FERMENTED WITH PENICILLIUM CANDIDUM ON ALZHEIMER’S DISEASE AND IDENTIFICATION OF A NOVEL OLEAMIDE WITH ENHANCED MICROGLIAL PHAGOCYTOSIS AND ANTI-INFLAMMATORY ACTIVITY
Yasuhisa Ano1, Makiko Ozawa2, Toshiko Kutsukake1, Shinya Sugiyama3, Kazuyuki Uchida2, Aruto Yoshida1, Hiroyuki Nakayama2, 1Kirin Company, Ltd., Kanagawa, Japan; 2The University of Tokyo, Tokyo, Japan; 3Koiwai Dairy Products Co., Ltd., Iwate, Japan. Contact e-mail: Yasuhisa_Ano@ kirin.co.jp Background: Despite the ever-increasing number of dementia patients worldwide, fundamental therapeutic approaches have not been established yet. Preventive measures to the disease in daily life including diet, nutrition, exercise, and learning activity continue to raise attention. Several epidemiological studies suggest that intake of fermented dairy products prevents cognitive decline in the elderly. However, the mechanism and the active compounds remain to be investigated. In this study we evaluated the preventive effects of fermented dairy products on dementia using Alzheimer’s disease model (5xFAD) animals with chronic inflammation and identified active compounds in dairy products. Methods: Camembert cheeses made by conventional fermentation with P. candidum weredelipidated and used as samples for diet preparation. Three-month old 5xFAD mice were fed diet with or without the samples (2% w/w) for three months. The mice were then sacrificed, and their b amyloid (Ab) accumulations, neurotrophic factors and cytokine productions were measured by ELISA and immunohistochemical methods. To identify the active component, primary microglia isolated from newborn murine brains were pretreated with fractionated sample extractions, and then treated with lipopolysaccharide and IFN-g for evaluation of cytokine productions and cell surface marker expressions by ELISA and flow cytometry, or treated with Ab-FAM for phagocytosis assay. Results: From among the cheeses examined, Camembert types fermented with P. candidum showed strikingly high anti-inflammatory activity. In the parallel-group study using 5xFAD mice, Ab accumulation and TNF-a and MIP-1a productions in the group fed the sample diet were significantly restrained. Production of hippocampal BDNF and GDNF and synaptophysin content increased in 5xFAD mice fed the samples. In addition, oleamide was identified as an active compound enhancing both micoglial phagocytosis and anti-inflammatory activity. Enhanced phagocytosis by oleamide was diminisched by PPAR-g antagonist treatment. Oleamide also induced M2 type CD206+CD11b+microglia and suppressed the production of TNF-a-and MIP-1a, and the expression of CD68, CD80 and CD86. Conclusions: The present study demonstrated the epidemiologically reported preventive effects of dairy products on Alzheimer’s disease. Oleamide identified as an active content in the P. candidum fermented dairy products reduces Ab accumulation and suppresses the induced chronic inflammation following Ab deposits, which might be helpful for the prevention of dementia.