- Email: [email protected]
Modulation of nitrergic pathway by sesamol prevents cognitive deficits and associated biochemical alterations in intracerebroventricular streptozotocin-administered rats
ICAD - Abstract Submission P3: Tuesday, July 19, 2011 Poster Presentations P3 immunoprecipitates. Results: We analyzed the cortical proteome of the APP23 mouse model, at presymptomatic (1 month) and symptomatic age (7 months). At presymptomatic age, proteins related to glycolysis were predominantly altered according to enrichment analysis. At symptomatic age, proteins related to oxidative phosphorylation were enriched. In addition to changes in energy metabolism, we observed an overall elevated protein oxidation status (Oxyblots) at presymptomatic and symptomatic age. Carbonylated proteins were identified by mass spectrometry. Conclusions: Enhanced ROS production in mitochondria caused by Abeta has been described previously. However, the production of ROS is an inevitable consequence of mitochondrial metabolism. A disturbed equilibrium between the production of ROS and antioxidant processes leads to enhanced ROS production which has widespread consequences through oxidative modification of proteins. Glycolytic enzymes, subunits of the mitochondrial ATPase complex and other energy metabolism associated proteins were identified as targets of oxidative modifications in AD patients. Interestingly, our OxyBlot analysis revealed enhanced carbonyl modifications of proteins in APP23 mice even at presymptomatic age. Our study provides evidence for a disequilibrium in the production and detoxification of reactive oxygen species in APP23 mice long before Abeta aggregation occurs. These data resemble observations in AD patients and underline the causative role of impaired energy metabolism in AD.
P3-182
GENETIC KNOCKOUT OF ESTROGEN RECEPTOR-€ıb¢ CONTRIBUTES TO ALZHEIMER’S DISEASE PATHOGENESIS BY IMPAIRING MITOCHONDRIAL FUNCTION IN FEMALES
Jiangang Long1, Yong Shen2, Rena Li3, 1Xi’an Jiaotong University School of Life Science and Technology, Xi An, China; 2Roskamp Institute, Center for Advanced Therapeutic Strategies for Brain Disorders, Sarasota, Florida, United States; 3Roskamp Institute, Sarasota, Florida, United States. Background: Mitochondria are the major source of energy for the normal functioning of brain cells. Increasing evidence suggests mitochondrial oxidative damage might be one of early events in Alzheimer’s disease (AD) progression. Estrogen has been shown to suppress mitochondrial oxidative stress, regulate energy metabolism, and regulate the expression of mitochondria-involved anti-apoptotic proteins like bcl-2 family and bcl-xL, which suggests that brain mitochondria may be major targets of estrogen action in the central nervous system. Studies also demonstrated that many of these protective effects can be blocked by an estrogen receptor (ER) antagonist, ICI-182780, suggesting an ER-mediated mode of estrogen action. Even more interestingly, recent reports have confirmed the localization of ERb in the mitochondria and suggested that estrogen can directly affect mitochondrial function through mitochondrial ERb (mtERb). Methods: To address whether female AD brains exhibit mitochondrial impairment and whether it is correlated with estrogen receptors, we conducted mitochondrial functional analyses in female AD patients and age-matched non-demented (ND) individuals. Using frontal cortex samples from the brains of age-matched AD and non-ND women, we examined mitochondrial cytochrome C oxidase (COX) activity, and reactive oxygen species (ROS)-induced intracellular protein oxidation as protein carbonylation along with receptor-b (ERb) sub cellular distribution and protein expression. In order to investigate whether lacking of ERb causes mitochondrial dysfunction, mice with ERb gene knockout were used as an animal model. Results: First, we found that the frontal cortices of female AD patients exhibited significantly reduced ERb expression, including in mitochondrial fraction, reduced mitochondrial COX activity, and increased protein carbonylation compared to the samples from ND. A significant linkage between the mitochondrial ERb expression and mitochondrial COX activity were observed in the female human brain, suggesting an involvement of ERb in mitochondrial function in the brain. Second, we noticed that mitochondria from the brains of ERb-knockout mice exhibited increased ROS generation and reduced mitochondrial membrane potential (MMP) under b-amyloid (Ab1-42 or Ab25-35) insult compared to brain mitochondria from wild-type control
S575
mice. These results suggest that ERb depletion can impair mitochondrial function in mice and may significantly contribute to the mitochondrial dysfunction involved in AD pathogenesis in women. Conclusions: Although further investigation is needed, our novel detection of ERb expression in neuronal mitochondria in the female human brain along with the direct correlation of mtERb expression with COX activity and the significant decrease of mtERb in the AD brain suggest a possible connection between ERb and mitochondrial dysfunction in AD. This hypothesis is supported by our experiments involving transgenic ERb knockout mice, which showed that ERb deficiency leads to mitochondrial dysfunction in the brain by decreasing MMP and by increasing mitochondrial vulnerability to Ab-induced ROS generation. Together, the results of this study indicate that ERb deficiency may play an important role in AD pathogenesis in females by contributing to mitochondrial dysfunction.
P3-183
ROLE OF RCAN1, A GENE INVOLVED IN THE ADAPTATION TO OXIDATIVE STRESS, IN ALZHEIMER’S PATHOLOGY
Ana Lloret1, Maria Carmen Badia2, Esther Giraldo1, Dolores Alonso3, Kelvin Davies4, Jose Vi~na1, 1University of Valencia, Valencia, Spain; 2 Hospital de Denia, denia, Alicante, Spain; 3Hospital Clinico Universitario, Valencia, Spain; 4University of Southern California, Los Angeles, California, United States. Background: Amyloid-b peptide toxicity and tau hyperphosphorylation are two major hallmarks of Alzheimer’s disease. However, their possible molecular relationship with regard to the pathophysiology of the disease has not been elucidated. Methods: Wistar rats were sacrificed at day 14 of gestation, and foetal neurons were isolated. After incubation with 5 micromolar beta-amyloid peptide, we mesured the expression of RCAN1, GSK3b and P-tau by western blotting. Silencing experiments were performed in PC12 cells. We also isolated lymphocytes from healthy persons carrying Apo E 3/4 or 4//4 alleles and measured RCAN1 and P-tau expression by western blotting. Results: We report that incubation of foetal rat cortical neurons with Ab up-regulates RCAN1 (regulator of calcineurin, a gene whose expression isup-regulated by oxidative stress). RCAN1 is a calcineurin inhibitor. Calcineurin itself is a phosphatase of phospho-tau. Activation of RCAN1 also causes an up-regulation of GSK3b (a tau phosphorylase). Incubation of neurons with Ab results in an increased phosphorylation of tau. All these changes do not take place when RCAN1 is silenced. Thus we propose a mechanism to link Ab toxicity and tau hyperphosphorylation: Ab causes mitochondrial oxidative stress and increases ROS production which results in anup-regulation of RCAN1. RCAN1 inhibitscalcineurin and also activates GSK3b. Both mechanisms shift tau to a hyperphosphorylated state. These results have a human correlation: lymphocytes from persons whose ApoE genotype is e4/e4 (with high risk of developing AD) show higher levels of RCAN1 and phospho-tau than those carrying the ApoE e3/e3 or e3/e4 genotypes. Conclusions: RCAN1 links betaamyloid toxicity with tau hyperphosphorilation via oxidative stress. RCAN1 levels are higher in lymphopcytes from persons carrying Apo E 4 allele. Thus up-regulation ofRCAN1 may be a marker of risk to develop Alzheimer’s disease.
P3-184
MODULATION OF NITRERGIC PATHWAY BY SESAMOL PREVENTS COGNITIVE DEFICITS AND ASSOCIATED BIOCHEMICAL ALTERATIONS IN INTRACEREBROVENTRICULAR STREPTOZOTOCIN-ADMINISTERED RATS
Shubham Misra1, Vinod Tiwari2, Anurag Kuhad3, Kanwaljit Chopra3, 1 University Institute of Pharmaceutical Sciences, Chandigarh, India; 2 Panjab University, Chandigarh, India; 3Univ Inst Pharm Sci, Panjab University, Chandigarh - 160 014 INDIA, Chandigarh, India. Background: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and widespread loss of neurons and their synapses in the cerebral cortex and hippocampus. Increasing
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ICAD - Abstract Submission P3: Tuesday, July 19, 2011 Poster Presentations P3
evidence indicates that factors such as oxidative-nitrergic stress, glutathione depletion, impaired protein metabolism and cholinergic deficit can interact in a vicious cycle, which is central to AD pathogenesis. Methods: Intracerebroventricular (i.c.v.) streptozotocininduced-cognitive impairment has been widely used as an experimental paradigm to study AD. Streptozotocin (3 mg/kg, i.c.v) was injected bilaterally in two divided doses on first and third day to rats making the dose of 1.5 mg/kg each day. Sesamol and different nitric oxide modulators were administered from day 0 to day 21. Results: In the present study, i.c.v. streptozotocin produced significant cognitive deficits as measured in Morris water maze and elevated plus maze task coupled with increased serum TNF-a levels and marked rise in brainacetylcholinesterase and oxidative-nitrergic stress in female Wistar rats. Administration of L-arginine (125 mg/kg i.p), anitric oxide donor, alone to i.c.v. streptozotocin treated rats accentuated behavioral and biochemical deficits and also abolished the protective effect of sesamol (8mg/kg). L-NAME (10 mg/kg i.p.), a non-specific NOS inhibitor significantly restored all the behavioral and biochemical indices in i.c.v. streptozotocin rats. Moreover, combination of L-NAME with sub-effective dose of sesamol (4 mg/kg) potentiated its protective effect. Conclusions: Our findings demonstrate the effectiveness of sesamol in preventing intracerebroventricular streptozotocin-induced cognitive deficits by modulating nitrergic signaling and oxido-inflammatory cascade. P3-185
A SYNTHETIC AMINO ACID SUBSTITUTION OF TYR10 IN ABETA PEPTIDE SEQUENCE ACTS AS A DOMINANT NEGATIVE IN AMYLOIDOGENESIS
Honoree Mazarguil1, Susanne Funke2, Dirk Bartnik3, Jacques Fantini4, Tiphany Gouget5, Dieter Willbold3, Michel Khrestchatisky6, Lorena Perrone6, 1IPBS, Toulouse, France; 2Forschungszentrum J€ulich, Juelich, Germany; 3Forschungszentrum J€ulich, J€ulich, Germany; 4CNRS UMR 6231, Marseille, France; 5NICN UMR6184, Marseiile, France; 6 NICN UMR6184, Marseille, France. Background: Alzheimer’s disease (AD) is the most common cause of dementia in elderly people and age is the major non-genetic risk factor for sporadic AD. A hallmark of AD is the accumulation of amyloid in the brain, which is composed mainly by the amyloid beta-peptide (Ab) in the form of oligomers and fibrils. However, how aging induces Ab aggregation is not yet fully determined. Tyr 10 in Ab sequences seems to be associated to the age-dependent risk factors for AD, such as (i) increased GM1 brain membrane content, (ii) altered lipid domain in brain membrane, (iii) oxidative stress. However, the role of Tyr 10 in Ab aggregation following interaction with the plasma membrane is not yet demonstrated. Methods: We decided to explore the function of Tyr10 in Ab aggregation in vitro and in cellulo. To elucidate the role of Tyr 10 in Ab aggregation and toxicity, we substituted in Ab1-42 sequence Tyr10 with the synthetic amino acid para-amino-Phenylalanine. We analyzed the aggregation properties of wild-type and mutant Ab in vitro and in cellulo. Moreover, we analyzed their uptake in neuronal cells. Results: The Ab mutant shows impaired aggregation properties and sensibly increased affinity for GM1 in vitro. It has a dominant negative effect on amyloidogenesis in vitro and in cellulo, where it impairs the aggregation of wild-type Ab. Moreover, this mutant does not induce oxidative stress and it blocks wildtype Ab -induced cell death. Conclusions: The present study shed new light in the understanding of Ab-membrane interactions in Ab-induced neurotoxicity. It demonstrates the relevance of Tyr 10 in (i) Ab-membrane interaction, (ii) Ab aggregation, (iii) Ab-induced oxidative stress. Furthermore, our results open the way for the design of peptides aimed to inhibit Ab aggregation and neurotoxicity. P3-186
THE EFFECT OF VPA IN PRESYMPTOMATIC ALZHEIMER’S DISEASE MODEL MICE
Hyemyung Seo1, Noh Haneul1, 1Hanyang University, Ansan, South Korea.
Background: Valproic acid (VPA), histone deacetylase inhibitor has been reported to enhance nuclear histone acetylation and neuronal viability. Tg 6799 mice over express mutant human APP (695) with the Swedish (K670N, M671L), Florida (I716V), and London (V717I) Familial Alzheimer’s disease (FAD) mutations and human PS1 harboring two FAD mutations, M146L and L286V. Methods: In the present study, we evaluated the effect of VPA in in vitro primary cortical cells from Tg6799 mice (E18). We also determined the effect of VPA on the survival and function of cholinergic neurons in the brain tissue regions and blood samples of Tg6799 mice at pre-symptomatic stage. To determine the biochemical changes in this AD transgenic model by VPA at pre-symptomatic stages, we dissected several brain regions and collected the blood from Tg6799 mice at 4 weeks and 16 weeks of age after VPA administration. We performed RT-PCR using RNA samples with matching primers for each cytokine gene and specific-marker protein. Results: We determined that the mRNA expression of several cytokines including NF-kB increased in blood samples of Tg6799 mice compared to littermate control mice. We also detected the expression levels of amyloid precursor protein (APP) processing related genes including full-size APP, secreted APP (APPs), and amyloid beta (Abeta), in blood samples of Tg6799 mice. VPA administration enhanced viability of cholinergic neurons in cortical primary culture. VPA altered the level of cytokines in blood and brain regions of Tg6799 mice. Conclusions: These data suggest that Tg6799 mice express the increased levels of mRNA of several cytokines and APP processing related products in blood. The level of cytokines and pathological marker proteins can be regulated by VPA treatment in Tg6799 mice at pre-symptomatic stage.
P3-187
OXIDATIVE STRESS, AMYLOID METABOLISM AND ALZHEIMER’S PATHOGENESIS: EXTRAPOLATION OF A CELL-BASED STUDY
Maitrayee Sinha1, 1Institute of Post Graduate Medical Edu. & Res. (IPGMER), Kolkata, India. Background: Sporadic Alzheimer’s disease (AD) is multifactorial in origin, but the key elements of the pathogenic process are mitochondrial dysfunction, oxidative stress, inflammatory response, protein aggregation. The molecular links among these potential pathogenic mechanisms are not fully elucidated, but oxidative stress because of its ability to alter cell signaling, gene expression, membrane properties, enzyme and receptor functions is most likely to influence the cross-talk among the probable pathogenic processes of AD. In an ongoing study in the lab we have observed that the age-related oxidative stress alters amyloid peptide metabolism in rat brain which is prevented by long-term dietary supplementation. This study has attempted to explore the role of oxidative stress in triggering and propagating amyloid peptide pathology in a cell based model. Methods: PC 12 cells have been subjected to an oxidative insult by exposure to H202 (100-400 mM) or t-butylhydroperoxide (100-400 mM) in the presence or absence of radical scavengers and antioxidants. The altered metabolism of amyloid peptide has been monitored by measuring the expression levels of APP, the intracellular and extra cellular level of Aß-42, the activities/ levels of ß secretase, d secretase as well as the degrading enzymes of Aß-42 namely neprilysin and insulin degrading enzyme (IDE). Oxidative damage markers, protein aggregation and cell death have also been measured in this experimental model. Results: Significant alterations in APP expression and intracellular content of Aß-42 have been observed along with accumulation of oxidative damage markers and conspicuous cell death in PC 12 cells exposed to oxidative stress. Conclusions: When extrapolated to in vivo situation, our results imply that oxidative stress present in aged brain can be a major factor in triggering and promoting altered APP metabolism leading to the genesis of sporadic AD. Conclusions: The study shows oxidative stress mediated Amyloid precursor protein metabolism alteration as also accumulation of Aß-42 in cell. This study has clear implications in understanding the common mechanisms of brain aging and AD.
P3-182
GENETIC KNOCKOUT OF ESTROGEN RECEPTOR-€ıb¢ CONTRIBUTES TO ALZHEIMER’S DISEASE PATHOGENESIS BY IMPAIRING MITOCHONDRIAL FUNCTION IN FEMALES
Jiangang Long1, Yong Shen2, Rena Li3, 1Xi’an Jiaotong University School of Life Science and Technology, Xi An, China; 2Roskamp Institute, Center for Advanced Therapeutic Strategies for Brain Disorders, Sarasota, Florida, United States; 3Roskamp Institute, Sarasota, Florida, United States. Background: Mitochondria are the major source of energy for the normal functioning of brain cells. Increasing evidence suggests mitochondrial oxidative damage might be one of early events in Alzheimer’s disease (AD) progression. Estrogen has been shown to suppress mitochondrial oxidative stress, regulate energy metabolism, and regulate the expression of mitochondria-involved anti-apoptotic proteins like bcl-2 family and bcl-xL, which suggests that brain mitochondria may be major targets of estrogen action in the central nervous system. Studies also demonstrated that many of these protective effects can be blocked by an estrogen receptor (ER) antagonist, ICI-182780, suggesting an ER-mediated mode of estrogen action. Even more interestingly, recent reports have confirmed the localization of ERb in the mitochondria and suggested that estrogen can directly affect mitochondrial function through mitochondrial ERb (mtERb). Methods: To address whether female AD brains exhibit mitochondrial impairment and whether it is correlated with estrogen receptors, we conducted mitochondrial functional analyses in female AD patients and age-matched non-demented (ND) individuals. Using frontal cortex samples from the brains of age-matched AD and non-ND women, we examined mitochondrial cytochrome C oxidase (COX) activity, and reactive oxygen species (ROS)-induced intracellular protein oxidation as protein carbonylation along with receptor-b (ERb) sub cellular distribution and protein expression. In order to investigate whether lacking of ERb causes mitochondrial dysfunction, mice with ERb gene knockout were used as an animal model. Results: First, we found that the frontal cortices of female AD patients exhibited significantly reduced ERb expression, including in mitochondrial fraction, reduced mitochondrial COX activity, and increased protein carbonylation compared to the samples from ND. A significant linkage between the mitochondrial ERb expression and mitochondrial COX activity were observed in the female human brain, suggesting an involvement of ERb in mitochondrial function in the brain. Second, we noticed that mitochondria from the brains of ERb-knockout mice exhibited increased ROS generation and reduced mitochondrial membrane potential (MMP) under b-amyloid (Ab1-42 or Ab25-35) insult compared to brain mitochondria from wild-type control
S575
mice. These results suggest that ERb depletion can impair mitochondrial function in mice and may significantly contribute to the mitochondrial dysfunction involved in AD pathogenesis in women. Conclusions: Although further investigation is needed, our novel detection of ERb expression in neuronal mitochondria in the female human brain along with the direct correlation of mtERb expression with COX activity and the significant decrease of mtERb in the AD brain suggest a possible connection between ERb and mitochondrial dysfunction in AD. This hypothesis is supported by our experiments involving transgenic ERb knockout mice, which showed that ERb deficiency leads to mitochondrial dysfunction in the brain by decreasing MMP and by increasing mitochondrial vulnerability to Ab-induced ROS generation. Together, the results of this study indicate that ERb deficiency may play an important role in AD pathogenesis in females by contributing to mitochondrial dysfunction.
P3-183
ROLE OF RCAN1, A GENE INVOLVED IN THE ADAPTATION TO OXIDATIVE STRESS, IN ALZHEIMER’S PATHOLOGY
Ana Lloret1, Maria Carmen Badia2, Esther Giraldo1, Dolores Alonso3, Kelvin Davies4, Jose Vi~na1, 1University of Valencia, Valencia, Spain; 2 Hospital de Denia, denia, Alicante, Spain; 3Hospital Clinico Universitario, Valencia, Spain; 4University of Southern California, Los Angeles, California, United States. Background: Amyloid-b peptide toxicity and tau hyperphosphorylation are two major hallmarks of Alzheimer’s disease. However, their possible molecular relationship with regard to the pathophysiology of the disease has not been elucidated. Methods: Wistar rats were sacrificed at day 14 of gestation, and foetal neurons were isolated. After incubation with 5 micromolar beta-amyloid peptide, we mesured the expression of RCAN1, GSK3b and P-tau by western blotting. Silencing experiments were performed in PC12 cells. We also isolated lymphocytes from healthy persons carrying Apo E 3/4 or 4//4 alleles and measured RCAN1 and P-tau expression by western blotting. Results: We report that incubation of foetal rat cortical neurons with Ab up-regulates RCAN1 (regulator of calcineurin, a gene whose expression isup-regulated by oxidative stress). RCAN1 is a calcineurin inhibitor. Calcineurin itself is a phosphatase of phospho-tau. Activation of RCAN1 also causes an up-regulation of GSK3b (a tau phosphorylase). Incubation of neurons with Ab results in an increased phosphorylation of tau. All these changes do not take place when RCAN1 is silenced. Thus we propose a mechanism to link Ab toxicity and tau hyperphosphorylation: Ab causes mitochondrial oxidative stress and increases ROS production which results in anup-regulation of RCAN1. RCAN1 inhibitscalcineurin and also activates GSK3b. Both mechanisms shift tau to a hyperphosphorylated state. These results have a human correlation: lymphocytes from persons whose ApoE genotype is e4/e4 (with high risk of developing AD) show higher levels of RCAN1 and phospho-tau than those carrying the ApoE e3/e3 or e3/e4 genotypes. Conclusions: RCAN1 links betaamyloid toxicity with tau hyperphosphorilation via oxidative stress. RCAN1 levels are higher in lymphopcytes from persons carrying Apo E 4 allele. Thus up-regulation ofRCAN1 may be a marker of risk to develop Alzheimer’s disease.
P3-184
MODULATION OF NITRERGIC PATHWAY BY SESAMOL PREVENTS COGNITIVE DEFICITS AND ASSOCIATED BIOCHEMICAL ALTERATIONS IN INTRACEREBROVENTRICULAR STREPTOZOTOCIN-ADMINISTERED RATS
Shubham Misra1, Vinod Tiwari2, Anurag Kuhad3, Kanwaljit Chopra3, 1 University Institute of Pharmaceutical Sciences, Chandigarh, India; 2 Panjab University, Chandigarh, India; 3Univ Inst Pharm Sci, Panjab University, Chandigarh - 160 014 INDIA, Chandigarh, India. Background: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and widespread loss of neurons and their synapses in the cerebral cortex and hippocampus. Increasing
S576
ICAD - Abstract Submission P3: Tuesday, July 19, 2011 Poster Presentations P3
evidence indicates that factors such as oxidative-nitrergic stress, glutathione depletion, impaired protein metabolism and cholinergic deficit can interact in a vicious cycle, which is central to AD pathogenesis. Methods: Intracerebroventricular (i.c.v.) streptozotocininduced-cognitive impairment has been widely used as an experimental paradigm to study AD. Streptozotocin (3 mg/kg, i.c.v) was injected bilaterally in two divided doses on first and third day to rats making the dose of 1.5 mg/kg each day. Sesamol and different nitric oxide modulators were administered from day 0 to day 21. Results: In the present study, i.c.v. streptozotocin produced significant cognitive deficits as measured in Morris water maze and elevated plus maze task coupled with increased serum TNF-a levels and marked rise in brainacetylcholinesterase and oxidative-nitrergic stress in female Wistar rats. Administration of L-arginine (125 mg/kg i.p), anitric oxide donor, alone to i.c.v. streptozotocin treated rats accentuated behavioral and biochemical deficits and also abolished the protective effect of sesamol (8mg/kg). L-NAME (10 mg/kg i.p.), a non-specific NOS inhibitor significantly restored all the behavioral and biochemical indices in i.c.v. streptozotocin rats. Moreover, combination of L-NAME with sub-effective dose of sesamol (4 mg/kg) potentiated its protective effect. Conclusions: Our findings demonstrate the effectiveness of sesamol in preventing intracerebroventricular streptozotocin-induced cognitive deficits by modulating nitrergic signaling and oxido-inflammatory cascade. P3-185
A SYNTHETIC AMINO ACID SUBSTITUTION OF TYR10 IN ABETA PEPTIDE SEQUENCE ACTS AS A DOMINANT NEGATIVE IN AMYLOIDOGENESIS
Honoree Mazarguil1, Susanne Funke2, Dirk Bartnik3, Jacques Fantini4, Tiphany Gouget5, Dieter Willbold3, Michel Khrestchatisky6, Lorena Perrone6, 1IPBS, Toulouse, France; 2Forschungszentrum J€ulich, Juelich, Germany; 3Forschungszentrum J€ulich, J€ulich, Germany; 4CNRS UMR 6231, Marseille, France; 5NICN UMR6184, Marseiile, France; 6 NICN UMR6184, Marseille, France. Background: Alzheimer’s disease (AD) is the most common cause of dementia in elderly people and age is the major non-genetic risk factor for sporadic AD. A hallmark of AD is the accumulation of amyloid in the brain, which is composed mainly by the amyloid beta-peptide (Ab) in the form of oligomers and fibrils. However, how aging induces Ab aggregation is not yet fully determined. Tyr 10 in Ab sequences seems to be associated to the age-dependent risk factors for AD, such as (i) increased GM1 brain membrane content, (ii) altered lipid domain in brain membrane, (iii) oxidative stress. However, the role of Tyr 10 in Ab aggregation following interaction with the plasma membrane is not yet demonstrated. Methods: We decided to explore the function of Tyr10 in Ab aggregation in vitro and in cellulo. To elucidate the role of Tyr 10 in Ab aggregation and toxicity, we substituted in Ab1-42 sequence Tyr10 with the synthetic amino acid para-amino-Phenylalanine. We analyzed the aggregation properties of wild-type and mutant Ab in vitro and in cellulo. Moreover, we analyzed their uptake in neuronal cells. Results: The Ab mutant shows impaired aggregation properties and sensibly increased affinity for GM1 in vitro. It has a dominant negative effect on amyloidogenesis in vitro and in cellulo, where it impairs the aggregation of wild-type Ab. Moreover, this mutant does not induce oxidative stress and it blocks wildtype Ab -induced cell death. Conclusions: The present study shed new light in the understanding of Ab-membrane interactions in Ab-induced neurotoxicity. It demonstrates the relevance of Tyr 10 in (i) Ab-membrane interaction, (ii) Ab aggregation, (iii) Ab-induced oxidative stress. Furthermore, our results open the way for the design of peptides aimed to inhibit Ab aggregation and neurotoxicity. P3-186
THE EFFECT OF VPA IN PRESYMPTOMATIC ALZHEIMER’S DISEASE MODEL MICE
Hyemyung Seo1, Noh Haneul1, 1Hanyang University, Ansan, South Korea.
Background: Valproic acid (VPA), histone deacetylase inhibitor has been reported to enhance nuclear histone acetylation and neuronal viability. Tg 6799 mice over express mutant human APP (695) with the Swedish (K670N, M671L), Florida (I716V), and London (V717I) Familial Alzheimer’s disease (FAD) mutations and human PS1 harboring two FAD mutations, M146L and L286V. Methods: In the present study, we evaluated the effect of VPA in in vitro primary cortical cells from Tg6799 mice (E18). We also determined the effect of VPA on the survival and function of cholinergic neurons in the brain tissue regions and blood samples of Tg6799 mice at pre-symptomatic stage. To determine the biochemical changes in this AD transgenic model by VPA at pre-symptomatic stages, we dissected several brain regions and collected the blood from Tg6799 mice at 4 weeks and 16 weeks of age after VPA administration. We performed RT-PCR using RNA samples with matching primers for each cytokine gene and specific-marker protein. Results: We determined that the mRNA expression of several cytokines including NF-kB increased in blood samples of Tg6799 mice compared to littermate control mice. We also detected the expression levels of amyloid precursor protein (APP) processing related genes including full-size APP, secreted APP (APPs), and amyloid beta (Abeta), in blood samples of Tg6799 mice. VPA administration enhanced viability of cholinergic neurons in cortical primary culture. VPA altered the level of cytokines in blood and brain regions of Tg6799 mice. Conclusions: These data suggest that Tg6799 mice express the increased levels of mRNA of several cytokines and APP processing related products in blood. The level of cytokines and pathological marker proteins can be regulated by VPA treatment in Tg6799 mice at pre-symptomatic stage.
P3-187
OXIDATIVE STRESS, AMYLOID METABOLISM AND ALZHEIMER’S PATHOGENESIS: EXTRAPOLATION OF A CELL-BASED STUDY
Maitrayee Sinha1, 1Institute of Post Graduate Medical Edu. & Res. (IPGMER), Kolkata, India. Background: Sporadic Alzheimer’s disease (AD) is multifactorial in origin, but the key elements of the pathogenic process are mitochondrial dysfunction, oxidative stress, inflammatory response, protein aggregation. The molecular links among these potential pathogenic mechanisms are not fully elucidated, but oxidative stress because of its ability to alter cell signaling, gene expression, membrane properties, enzyme and receptor functions is most likely to influence the cross-talk among the probable pathogenic processes of AD. In an ongoing study in the lab we have observed that the age-related oxidative stress alters amyloid peptide metabolism in rat brain which is prevented by long-term dietary supplementation. This study has attempted to explore the role of oxidative stress in triggering and propagating amyloid peptide pathology in a cell based model. Methods: PC 12 cells have been subjected to an oxidative insult by exposure to H202 (100-400 mM) or t-butylhydroperoxide (100-400 mM) in the presence or absence of radical scavengers and antioxidants. The altered metabolism of amyloid peptide has been monitored by measuring the expression levels of APP, the intracellular and extra cellular level of Aß-42, the activities/ levels of ß secretase, d secretase as well as the degrading enzymes of Aß-42 namely neprilysin and insulin degrading enzyme (IDE). Oxidative damage markers, protein aggregation and cell death have also been measured in this experimental model. Results: Significant alterations in APP expression and intracellular content of Aß-42 have been observed along with accumulation of oxidative damage markers and conspicuous cell death in PC 12 cells exposed to oxidative stress. Conclusions: When extrapolated to in vivo situation, our results imply that oxidative stress present in aged brain can be a major factor in triggering and promoting altered APP metabolism leading to the genesis of sporadic AD. Conclusions: The study shows oxidative stress mediated Amyloid precursor protein metabolism alteration as also accumulation of Aß-42 in cell. This study has clear implications in understanding the common mechanisms of brain aging and AD.