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Acute inhibition of gamma secretase lowers abeta dimer levels in plaque-bearing PSAPP mice
Poster Presentations P2 pathology appears to be due to LC degeneration and not simply low NA levels. This suggests that NA potentiates Ab toxicity. Indeed, catecholamines potentiate Ab misfolding and oxidative toxicity. For example, catecholamines both undergo autooxidation and increase Ab oxidative stress. Dopamine, a catecholamine, increases Ab misfolding into toxic oligomeric amyloid forms and it is likely that other catecholamines have a similar effect. Currently, the mechanism of how catecholamines catalyze Ab misfolding and augment oxidative toxicity is unknown. Methods: We have studied the effects of the catecholamines, dopamine, noradrenaline and adrenaline on Ab misfolding and oxidative stress in vitro using mass spectrometry, western blots, electron microscopy, protease protections assays and cell culture. Results: Our data demonstrates that all catecholamines increase Ab misfolding (likely oligomers) in vitro. Preliminary protease protection assay experiments and electron microscopy suggest that Ab oligomers are formed upon incubation with catecholamines. Additionally, we demonstrate that this process is enhanced by addition of the transition metal Cu2+ and inhibited by EDTA and the antioxidant BHT. Structurally related compounds incompetent for autooxidation do not cause Ab misfolding or oxidative stress. The extent of catecholamine oxidation will be ascertained via LC/ MS and adduct formation with Ab via MALDI. Additionally, the effect of catecholamines on oxidative toxicity in vitro and in cultured cells will be ascertained. Conclusions: Thus, we have evidence that catecholamine neurotransmitters catalyze Ab misfolding and augment its oxidative stress in vitro. Extrapolation from this data suggests that catecholamines could play a role in Ab pathogenesis in AD.
P2-254
ACUTE INHIBITION OF GAMMA SECRETASE LOWERS ABETA DIMER LEVELS IN PLAQUEBEARING PSAPP MICE
Elie Needle, Kevin Atchison, Charles Nolan, Ashley Robshaw, Nathalie Breysse, Shi Liang, Yi Chen, Cathleen Gonzales, Margaret Zaleska, David Riddell, Warren Hirst, Pfizer, Groton, Connecticut, United States. Background: Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that is characterized by elevated levels of the amyloid beta-protein (Abeta). A number of studies have described the biochemical characterization of Abeta extracted from AD brain, demonstrating the presence of monomer and sodium dodecylsulfate (SDS)-stable dimers in both the watersoluble and formic acid-soluble fractions of brain. Objective: Our previous results have demonstrated SDS stable dimers appear in aged PSAPP mice together with plaque, electrophysiological and behavioral deficits. In the current study we investigated whether the source of dimeric Abeta was from the accumulated plaque or whether it was from a pool of newly synthesized Abeta. Methods: Plaque-bearing PSAPP (Tg2576/PS1M146L) mice were acutely treated with a high dose gamma-secretase inhibitor to block Abeta biosynthesis. Eight hours later, soluble Abeta oligomers were measured from the brains of these mice using a sensitive immunoprecipitation/Western blotting protocol after homogenizing the whole cerebra in Tris-buffered saline (TBS). Total, Abeta40 and Abeta42 levels were additionally measured by specific ELISA. Results: The 6 month old PSAPP mice are characterized as having an increasing plaque load, yet no behavioral deficits as observed by the Morris Water Maze. When acutely treated with a gamma-secretase inhibitor, we observed a 65% lowering of TBS soluble SDS stable dimers. Similar reductions in Abeta monomers were observed. We also detected a marked accumulation of the beta-C-terminal fragment (beta-CTF) in the TBS-Triton (membrane) extract, consistent with gamma-secretase inhibition. In the 12-month-old mice the gamma-secretase inhibitor reduced dimer by 47%. Total Abeta and specific Abeta40 and Abeta42 measurements by ELISA demonstrated similar Abeta lowering in the soluble (TBS) extracts, but not in the guanidine HCl extracts. Conclusions: These data support the use of the PSAPP mouse model system
S393
to test in vivo effects of inhibitors which target the APP processing cascade. We have demonstrated that a significant amount of dimeric Abeta is a pool of secretase derived, newly-synthesized Abeta and not derived from plaques. The ability to assess the biochemistry of these processes in vivo will allow for a correlation of physiological or behavioral effects in future studies.
P2-255
MICROGLIAL RAGE-DEPENDENT SIGNAL PATHWAY CONTRIBUTES TO SYNAPTIC DYSFUNCTION INDUCED BY AMYLOID AND TRANSIENT ISCHEMIA IN ENTORHINAL CORTEX
Nicola Origlia1, Ottavio Arancio2, Shi Du Yan2, Luciano Domenici1, 1 CNR, Pisa, Italy; 2Columbia University, New York, New York, United States. Background: Hypoxic condition may be the cause of progressive neuronal alterations in Alzheimer’s disease (AD). We raised the hypothesis that transient ischemia activates the Receptor for Advanced Glycation End products (RAGE) in different cell targets, resulting in synaptic dysfunction. RAGE is a cell surface binding site for several ligands, including beta-amyloid (Aß), and its expression is altered in neuronal and non-neuronal cells under ischemic conditions. Methods: We evaluated the effect of transient ischemia on field potentials (fEPSP) recorded in enthorinal cortex (EC) slices. We exposed EC slices to oxygen-glucose deprivation (OGD) for 10 min. We investigated the effects of OGD on synaptic transmission in: i) EC slices treated with blocking antibodies against RAGE, ii) EC slices from RAGE null mutant (RAGE-/-) or transgenic (Tg) mice in which defective RAGE is expressed selectively in neurons (DN-RAGE) or in microglia (DNMSR). To test whether Aß enriched environment increases synaptic dysfunction the effect of acute OGD on neuronal impairment was evaluated in slices treated with amyloid peptide (Aß42) or in slices from Tg APPsweInd mice. Results: In EC slices OGD caused depression of fEPSPs that slowly recover after re-introduction of oxygenated ACSF resulting in steady-state synaptic depression (81 6 4 % of baseline, n ¼ 13). Absence of RAGE in EC slices from RAGE (-/-) mice resulted in complete recovery of fEPSPs amplitude following OGD (99 6 6% of baseline; n ¼ 6, p < 0.05 vs. OGD in wt). The same protective effect was achieved in EC slices from Tg DNMSR mice but not in Tg DN-RAGE slices. Interestingly, blocking inflammatory cytokine IL-1beta signaling or inhibition of stress activated kinases (p38 MAPK and JNK) prevented OGD-dependent synaptic dysfunction. Moreover, we found that the effects of OGD were enhanced in Aß perfused or tg APP slices. Importantly, the absence of RAGE or selective deficiency of RAGE in microglia (DNMSR) protected from synaptic impairment induced by Aß coupled to OGD. Conclusions: Our results indicate cell-specific contribution of RAGE to the effects of ischemia on synaptic function in Aß enriched environment. We suggest that RAGE may be viewed as part of a neural system that controls synaptic responses in vascular pathology and AD.
P2-256
HISTOLOGICAL LOCALIZATION OF AMYLOIDb OLIGOMERS IN THE HUMAN BRAIN DURING THE COURSE OF ALZHEIMER’S DISEASE
Ma€ı Panchal1, Pascale Lacor2, William Klein2, Charles Duyckaerts1, 1 H^opital de la Salp^etriere, CRICM, Paris, France; 2Northwestern University, Evanston, Illinois, United States. Background: It is generally admitted that soluble Abeta oligomers are the toxic species in Alzheimer’s disease (AD) while fibrils seem devoid of effects. Synthetic Abeta oligomers or isolated from AD brain homogenates bind to synapses in vitro and disrupt their morphology and function. It has
P2-254
ACUTE INHIBITION OF GAMMA SECRETASE LOWERS ABETA DIMER LEVELS IN PLAQUEBEARING PSAPP MICE
Elie Needle, Kevin Atchison, Charles Nolan, Ashley Robshaw, Nathalie Breysse, Shi Liang, Yi Chen, Cathleen Gonzales, Margaret Zaleska, David Riddell, Warren Hirst, Pfizer, Groton, Connecticut, United States. Background: Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that is characterized by elevated levels of the amyloid beta-protein (Abeta). A number of studies have described the biochemical characterization of Abeta extracted from AD brain, demonstrating the presence of monomer and sodium dodecylsulfate (SDS)-stable dimers in both the watersoluble and formic acid-soluble fractions of brain. Objective: Our previous results have demonstrated SDS stable dimers appear in aged PSAPP mice together with plaque, electrophysiological and behavioral deficits. In the current study we investigated whether the source of dimeric Abeta was from the accumulated plaque or whether it was from a pool of newly synthesized Abeta. Methods: Plaque-bearing PSAPP (Tg2576/PS1M146L) mice were acutely treated with a high dose gamma-secretase inhibitor to block Abeta biosynthesis. Eight hours later, soluble Abeta oligomers were measured from the brains of these mice using a sensitive immunoprecipitation/Western blotting protocol after homogenizing the whole cerebra in Tris-buffered saline (TBS). Total, Abeta40 and Abeta42 levels were additionally measured by specific ELISA. Results: The 6 month old PSAPP mice are characterized as having an increasing plaque load, yet no behavioral deficits as observed by the Morris Water Maze. When acutely treated with a gamma-secretase inhibitor, we observed a 65% lowering of TBS soluble SDS stable dimers. Similar reductions in Abeta monomers were observed. We also detected a marked accumulation of the beta-C-terminal fragment (beta-CTF) in the TBS-Triton (membrane) extract, consistent with gamma-secretase inhibition. In the 12-month-old mice the gamma-secretase inhibitor reduced dimer by 47%. Total Abeta and specific Abeta40 and Abeta42 measurements by ELISA demonstrated similar Abeta lowering in the soluble (TBS) extracts, but not in the guanidine HCl extracts. Conclusions: These data support the use of the PSAPP mouse model system
S393
to test in vivo effects of inhibitors which target the APP processing cascade. We have demonstrated that a significant amount of dimeric Abeta is a pool of secretase derived, newly-synthesized Abeta and not derived from plaques. The ability to assess the biochemistry of these processes in vivo will allow for a correlation of physiological or behavioral effects in future studies.
P2-255
MICROGLIAL RAGE-DEPENDENT SIGNAL PATHWAY CONTRIBUTES TO SYNAPTIC DYSFUNCTION INDUCED BY AMYLOID AND TRANSIENT ISCHEMIA IN ENTORHINAL CORTEX
Nicola Origlia1, Ottavio Arancio2, Shi Du Yan2, Luciano Domenici1, 1 CNR, Pisa, Italy; 2Columbia University, New York, New York, United States. Background: Hypoxic condition may be the cause of progressive neuronal alterations in Alzheimer’s disease (AD). We raised the hypothesis that transient ischemia activates the Receptor for Advanced Glycation End products (RAGE) in different cell targets, resulting in synaptic dysfunction. RAGE is a cell surface binding site for several ligands, including beta-amyloid (Aß), and its expression is altered in neuronal and non-neuronal cells under ischemic conditions. Methods: We evaluated the effect of transient ischemia on field potentials (fEPSP) recorded in enthorinal cortex (EC) slices. We exposed EC slices to oxygen-glucose deprivation (OGD) for 10 min. We investigated the effects of OGD on synaptic transmission in: i) EC slices treated with blocking antibodies against RAGE, ii) EC slices from RAGE null mutant (RAGE-/-) or transgenic (Tg) mice in which defective RAGE is expressed selectively in neurons (DN-RAGE) or in microglia (DNMSR). To test whether Aß enriched environment increases synaptic dysfunction the effect of acute OGD on neuronal impairment was evaluated in slices treated with amyloid peptide (Aß42) or in slices from Tg APPsweInd mice. Results: In EC slices OGD caused depression of fEPSPs that slowly recover after re-introduction of oxygenated ACSF resulting in steady-state synaptic depression (81 6 4 % of baseline, n ¼ 13). Absence of RAGE in EC slices from RAGE (-/-) mice resulted in complete recovery of fEPSPs amplitude following OGD (99 6 6% of baseline; n ¼ 6, p < 0.05 vs. OGD in wt). The same protective effect was achieved in EC slices from Tg DNMSR mice but not in Tg DN-RAGE slices. Interestingly, blocking inflammatory cytokine IL-1beta signaling or inhibition of stress activated kinases (p38 MAPK and JNK) prevented OGD-dependent synaptic dysfunction. Moreover, we found that the effects of OGD were enhanced in Aß perfused or tg APP slices. Importantly, the absence of RAGE or selective deficiency of RAGE in microglia (DNMSR) protected from synaptic impairment induced by Aß coupled to OGD. Conclusions: Our results indicate cell-specific contribution of RAGE to the effects of ischemia on synaptic function in Aß enriched environment. We suggest that RAGE may be viewed as part of a neural system that controls synaptic responses in vascular pathology and AD.
P2-256
HISTOLOGICAL LOCALIZATION OF AMYLOIDb OLIGOMERS IN THE HUMAN BRAIN DURING THE COURSE OF ALZHEIMER’S DISEASE
Ma€ı Panchal1, Pascale Lacor2, William Klein2, Charles Duyckaerts1, 1 H^opital de la Salp^etriere, CRICM, Paris, France; 2Northwestern University, Evanston, Illinois, United States. Background: It is generally admitted that soluble Abeta oligomers are the toxic species in Alzheimer’s disease (AD) while fibrils seem devoid of effects. Synthetic Abeta oligomers or isolated from AD brain homogenates bind to synapses in vitro and disrupt their morphology and function. It has