- Email: [email protected]
SYNTHETIC β-AMYLOID DIMER IN ANTI-PARALLEL CONFORMATION INDUCES IN VIVO SYNAPTIC PLASTICITY DEFICITS
P870
Poster Presentations: P4
a secretory peptide which can inhibit the neurotoxicity of Ab. However, the neuroprotective effects and mechanism(s) of HN on Ab-induced AD-like pathological changes and memory deficits are yet incompletely defined. Methods: The animal models were established by injected of 2 ml Ab 42 into the hippocampus CA1. HN-treatment group was given 2 ml HN at the same site after the success of model making. Spatial memory was measured by Morris water maze test on the 7th day after injection. The dendritic morphology and dendritic spine density of the pyramidal neurons in the hippocampal CA1 of the rat was analyzed by the Golgi staining and the synaptic transmission was detected by electrophysiological recording. The expressions of several memory-associated proteins were detected by Western blotting, and the ultrastructure of neurons in hippocampal CA1 region of the rats was studied by transmission electron microscope. Results: In the present study, supplementation of HN has been approved to enhance long term potential and ameliorate memory deficits induced by Ab42, with increase dendritic branches and the density of the dendritic spines, and upregulation of pre- and post-synaptic protein levels. Supplementation of HN also attenuated the Ab42-induced tau hyperphosphorylation through activation protein phosphatase-2A (PP2A) with decreased inhibitory phosphorylated PP2A catalytic subunit (PP2AC) at Tyr307. At last, we also found that supplementation of HN ameliorated the Ab42-induced apoptosis and oxidative stress. Conclusions: The observations, in all, reveal the important roles of HN against Ab 42 -induced AD-like pathological changes and memory deficits.
P4-225
SYNTHETIC b-AMYLOID DIMER IN ANTIPARALLEL CONFORMATION INDUCES IN VIVO SYNAPTIC PLASTICITY DEFICITS
Hye Yun Kim, Hyunjin Vincent Kim, Sejin Lee, Dong Jin Kim, Young Soo Kim, Korea Institute of Science and Technology, Seoul, South Korea. Contact e-mail: [email protected] Background: Soluble Ab dimers are considered to play a critical role in Alzheimer’s disease. Despite many efforts to investigate Ab dimers via isolation or chemical synthesis, structural understanding of the dimeric conformation is unclear. As the smallest oligomeric species, Ab dimers, directly extracted from the cerebral cortex of AD patients, are found to impair synaptic plasticity and memory in rodents. Accordingly, substantial efforts have been devoted to understanding the pathophysiological function of Ab dimers by chemical approaches. The synthesis of a disulfide-linked Ab (1-40S26C) dimer enabled generation of anti-dimer monoclonal antibodies to neutralize synaptic plasticity. As Ab monomers are essential for brain function, selectively targeting pathogenic dimers is a considerable therapeutic and diagnostic approach for AD. Although covalently linked Ab dimers have given freedom from concerns of unstable oligomerization throughout biological studies, conformational relativity of full-length Ab dimers to AD pathogenesis in vivo is yet to be discovered. Here we explore the pathologically favored dimeric conformation using synthetic parallel and anti-parallel Ab (1-40) dimers with flexible linkers to facilitate dimerization. Methods: We synthesized Ab dimers in parallel and anti-parallel conformations by modified stepwise Fmoc solid-phase peptide synthesis protocols. To investigate if our dimer models facilitate interactions between linked Ab monomer strands, we performed simulations of molecular dynamics. We assessed amyloidogenic properties of the synthetic Ab dimers in a time-dependent manner using thioflavin-T assay, electrophoresis and transmission electron microscopy. At last, we induced AD-like behaviors in ICR mice by direct infusion of Ab peptides into the intracerebroventricular region and performed Y-maze / passive avoidance tests. Results: Of the two constructs, the anti-parallel dimer showed substantial development of oligomers and fibrils. When administered by intracerebroventricular injection, the anti-parallel dimer induced hippocampus-dependent learning and memory deficits in normal mice, similar to the monomer with doubled concentration. Our data place the anti-parallel conformation as the neurotoxic
and amyloidogenic structure of Ab dimers. Conclusions: Our study supports the view that pathogenic Ab assembly involves b-sheet-rich ordered dimer rather than disordered aggregates. Overall, our results suggest that majority of Ab monomers may aggregate into anti-parallel dimeric conformation during development of AD.
P4-226
ROLE OF PURINERGIC RECEPTORS IN ALZHEIMER’S DISEASE: INSIGHTS FROM ANIMAL MODELS
Majid Amar, Ihsen Youssef, Elodie Martin, Beno^ıt Delatour, Cecile Delarasse, Brain and Spine Institute (ICM), Paris, France. Contact e-mail: [email protected] Background: Purinergic P2X7 receptors (P2X7R) have been linked to the physiopathology of Alzheimer’s disease (AD) as their expression is significantly increased in glial cells populations surrounding amyloid plaques in human brain tissues and also in tissues from mice models of AD. It is our hypothesis that P2X7R may actually act as a "double-edged sword", (1) by activating non-amyloidogenic pathways and promoting neurotrophic effects ; we hence recently demonstrated in vitro that stimulation of P2X7R increases levels of sAPPa, (2) by inducing a neuroinflammatory response with deleterious effects through uncontrolled microglial activation at later stages of the disease. Methods: APPxPS1 transgenic mice modeling AD brain lesions were crossed with P2X7RKO mice to generate AD transgenics with or without functional P2X7R. Aged animals (>10 months) with fullblown pathology were behaviorally assessed in different learning & memory paradigms (short- and long-term spatial memories, executive functions) before being sacrificed. Histological (assessment of amyloid loads, evaluation of inflammation), biochemical (ELISA dosage of cytokines and chemokines) and flow cytometry analysis (phenotyping of microglial cells) were subsequently performed. Results: We first evidenced that invalidation of P2X7R induced a significant recovery of function in APPxPS1 mice trained in a cognitive paradigm. In parallel we showed, in the same animals, a drastic (-40%) reduction of amyloid loads (% of tissue occupied by plaques) in APPxPS1xP2X7RKO as compared to APPxPS1 transgenics. Decrease in plaques loads was observed in the isocortex, in the hippocampus and in subcortical (thalamic) regions. Examination of neuroinflammatory profiles in the different groups is currently under progress. Conclusions: While we previously showed a neuroprotective effect of P2X7R stimulation, the present data indicate that, at late stages of the disease, P2X7R exerts deleterious functions and potentiates AD-related lesions and symptoms. This strengthens the status of P2X7R as a target for future pharmacological intervention.
Poster Presentations: P4
a secretory peptide which can inhibit the neurotoxicity of Ab. However, the neuroprotective effects and mechanism(s) of HN on Ab-induced AD-like pathological changes and memory deficits are yet incompletely defined. Methods: The animal models were established by injected of 2 ml Ab 42 into the hippocampus CA1. HN-treatment group was given 2 ml HN at the same site after the success of model making. Spatial memory was measured by Morris water maze test on the 7th day after injection. The dendritic morphology and dendritic spine density of the pyramidal neurons in the hippocampal CA1 of the rat was analyzed by the Golgi staining and the synaptic transmission was detected by electrophysiological recording. The expressions of several memory-associated proteins were detected by Western blotting, and the ultrastructure of neurons in hippocampal CA1 region of the rats was studied by transmission electron microscope. Results: In the present study, supplementation of HN has been approved to enhance long term potential and ameliorate memory deficits induced by Ab42, with increase dendritic branches and the density of the dendritic spines, and upregulation of pre- and post-synaptic protein levels. Supplementation of HN also attenuated the Ab42-induced tau hyperphosphorylation through activation protein phosphatase-2A (PP2A) with decreased inhibitory phosphorylated PP2A catalytic subunit (PP2AC) at Tyr307. At last, we also found that supplementation of HN ameliorated the Ab42-induced apoptosis and oxidative stress. Conclusions: The observations, in all, reveal the important roles of HN against Ab 42 -induced AD-like pathological changes and memory deficits.
P4-225
SYNTHETIC b-AMYLOID DIMER IN ANTIPARALLEL CONFORMATION INDUCES IN VIVO SYNAPTIC PLASTICITY DEFICITS
Hye Yun Kim, Hyunjin Vincent Kim, Sejin Lee, Dong Jin Kim, Young Soo Kim, Korea Institute of Science and Technology, Seoul, South Korea. Contact e-mail: [email protected] Background: Soluble Ab dimers are considered to play a critical role in Alzheimer’s disease. Despite many efforts to investigate Ab dimers via isolation or chemical synthesis, structural understanding of the dimeric conformation is unclear. As the smallest oligomeric species, Ab dimers, directly extracted from the cerebral cortex of AD patients, are found to impair synaptic plasticity and memory in rodents. Accordingly, substantial efforts have been devoted to understanding the pathophysiological function of Ab dimers by chemical approaches. The synthesis of a disulfide-linked Ab (1-40S26C) dimer enabled generation of anti-dimer monoclonal antibodies to neutralize synaptic plasticity. As Ab monomers are essential for brain function, selectively targeting pathogenic dimers is a considerable therapeutic and diagnostic approach for AD. Although covalently linked Ab dimers have given freedom from concerns of unstable oligomerization throughout biological studies, conformational relativity of full-length Ab dimers to AD pathogenesis in vivo is yet to be discovered. Here we explore the pathologically favored dimeric conformation using synthetic parallel and anti-parallel Ab (1-40) dimers with flexible linkers to facilitate dimerization. Methods: We synthesized Ab dimers in parallel and anti-parallel conformations by modified stepwise Fmoc solid-phase peptide synthesis protocols. To investigate if our dimer models facilitate interactions between linked Ab monomer strands, we performed simulations of molecular dynamics. We assessed amyloidogenic properties of the synthetic Ab dimers in a time-dependent manner using thioflavin-T assay, electrophoresis and transmission electron microscopy. At last, we induced AD-like behaviors in ICR mice by direct infusion of Ab peptides into the intracerebroventricular region and performed Y-maze / passive avoidance tests. Results: Of the two constructs, the anti-parallel dimer showed substantial development of oligomers and fibrils. When administered by intracerebroventricular injection, the anti-parallel dimer induced hippocampus-dependent learning and memory deficits in normal mice, similar to the monomer with doubled concentration. Our data place the anti-parallel conformation as the neurotoxic
and amyloidogenic structure of Ab dimers. Conclusions: Our study supports the view that pathogenic Ab assembly involves b-sheet-rich ordered dimer rather than disordered aggregates. Overall, our results suggest that majority of Ab monomers may aggregate into anti-parallel dimeric conformation during development of AD.
P4-226
ROLE OF PURINERGIC RECEPTORS IN ALZHEIMER’S DISEASE: INSIGHTS FROM ANIMAL MODELS
Majid Amar, Ihsen Youssef, Elodie Martin, Beno^ıt Delatour, Cecile Delarasse, Brain and Spine Institute (ICM), Paris, France. Contact e-mail: [email protected] Background: Purinergic P2X7 receptors (P2X7R) have been linked to the physiopathology of Alzheimer’s disease (AD) as their expression is significantly increased in glial cells populations surrounding amyloid plaques in human brain tissues and also in tissues from mice models of AD. It is our hypothesis that P2X7R may actually act as a "double-edged sword", (1) by activating non-amyloidogenic pathways and promoting neurotrophic effects ; we hence recently demonstrated in vitro that stimulation of P2X7R increases levels of sAPPa, (2) by inducing a neuroinflammatory response with deleterious effects through uncontrolled microglial activation at later stages of the disease. Methods: APPxPS1 transgenic mice modeling AD brain lesions were crossed with P2X7RKO mice to generate AD transgenics with or without functional P2X7R. Aged animals (>10 months) with fullblown pathology were behaviorally assessed in different learning & memory paradigms (short- and long-term spatial memories, executive functions) before being sacrificed. Histological (assessment of amyloid loads, evaluation of inflammation), biochemical (ELISA dosage of cytokines and chemokines) and flow cytometry analysis (phenotyping of microglial cells) were subsequently performed. Results: We first evidenced that invalidation of P2X7R induced a significant recovery of function in APPxPS1 mice trained in a cognitive paradigm. In parallel we showed, in the same animals, a drastic (-40%) reduction of amyloid loads (% of tissue occupied by plaques) in APPxPS1xP2X7RKO as compared to APPxPS1 transgenics. Decrease in plaques loads was observed in the isocortex, in the hippocampus and in subcortical (thalamic) regions. Examination of neuroinflammatory profiles in the different groups is currently under progress. Conclusions: While we previously showed a neuroprotective effect of P2X7R stimulation, the present data indicate that, at late stages of the disease, P2X7R exerts deleterious functions and potentiates AD-related lesions and symptoms. This strengthens the status of P2X7R as a target for future pharmacological intervention.