- Email: [email protected]
PS1 Alzheimer mice
S248
Poster Presentations P1
as the Ab42/Ab40 ratio, were increased in Purkinje neurons from sporadic AD cases as compared to controls. However, the levels of Ab42 as well as Ab40 were clearly lower in Purkinje neurons than in pyramidal neurons. Based on the volume of the captured neurons the intraneuronal molar concentrations of Ab were calculated. Conclusions: We suggest that the concentration of intracellular Ab42 correlates to the neuropathology of Alzheimer’s disease. P1-257
IMPACT OF CHOLESTEROL ON APP DIMERS’ FORMATION, USING FRET DETECTION AND FLUORESCENCE DECAY IMAGING
Viviane Devauges1,2, Catherine Marquer3, Ge´raldine Liot4, Pierre Blandin1,2, Jack-Christophe Cossec3, Sandrine Le´cart5, Sandrine Humbert4, Fre´de´ric Saudou4, Fre´de´ric Druon2,5, Patrick Georges2,5, Marie-Claude Potier3, Sandrine Le´veˆque-Fort1,5, 1Laboratoire de Photophysique Mole´culaire, Orsay, France; 2Laboratoire Charles Fabry de l’Institut d’Optique, Palaiseau, France; 3CRICM/UPMC, Paris, France; 4Institut Curie, Orsay, France; 5Centre de Photonique Biome´dicale, Orsay, France. Contact e-mail: [email protected] Background: One of the major hallmarks of Alzheimer’s disease (AD) is the presence in the brain of senile plaques, mainly composed of Ab peptide. It results mainly from the sequential cleavage of APP (amyloid precursor protein) by the b-secretase BACE1. Though it seems clear that APP can dimerize, the sequence implied in the dimerization process and the impact of APP dimerization on Ab secretion is still under debate. Recent genetic, epidemiologic and biochemical data point to a link between cholesterol and AD. We previously showed that increasing plasma membrane cholesterol content leads to a gain in APP-BACE1 proximity at the plasma membrane, increased APP endocytosis and a higher Ab production. We thus wondered how increasing membrane cholesterol would impact on APP dimerization. Methods: Two different FRET methods were developped to assess APP dimerization in living cells. Firstly, proximity (<10 nm) of APP-GFP and APP-mCherry was monitored by FRET-FLIM (Fluorescence Lifetime Imaging to monitor GFP’s lifetime). Then, homo-FRET between GFPs was followed using time-resolved fluorescence anisotropy imaging of cells expressing APP-GFP. These experiments were performed under both TIRF (plasma membrane only) and epifluorescence (whole cell) illumination. Modulation of membrane cholesterol was performed in HEK-293 cell lines and embryonic rat hippocampal neurons using a MbCD-cholesterol complex and was kinetically followed over 30 minutes. Results: First measurements were made with cytosolic GFP or with a membrane anchored GFPfusion protein to ensure that increasing cholesterol concentration at the plasma membrane of cells does not have an impact on GFP anisotropy on its own. Thus the effects observed on APP-GFP when adding cholesterol are specifically due to homo-FRET. We used constituvely dimerizing mutant APP as a positive control. We are currently investigating the effect of cholesterol on APP dimerization using FRET-FLIM and homo-FRET on both cell types. Comparative results will be presented. Conclusions: We developed two novel complementary FRET techniques to probe APP dimerization in living cells. They were used to unravel the role of cholesterol on the formation of APP dimers in HEK-293 cells and in neurons, thus giving new insight on a possible link between APP dimerization and Ab production. P1-258
ABCA1 MEDIATES THE BENEFICIAL EFFECTS OF THE LIVER-X-RECEPTOR AGONIST GW3965 ON AMYLOID LOAD AND OBJECT RECOGNITION MEMORY IN APP/PS1 ALZHEIMER MICE
Cheryl L. Wellington1, James Donkin1, Sophie Stukas1, Veronica HirschReinshagen1, Dhananjay Namjoshi1, Anna Wilkinson1, Sharon May1, Jennifer Chan1, Jianjia Fan1, Jon Collins2, 1University of British Columbia, Vancouver, BC, Canada; 2GlaxoSmithKline, Research Triangle Park, NC, USA. Contact e-mail: [email protected] Background: Apolipoprotein E (apoE) is the major cholesterol carrier in the brain and the most validated genetic risk factor for Alzheimer’s Disease
(AD), the leading cause of dementia in the elderly characterized by Ab and amyloid deposits in brain tissue. We have shown that the cholesterol transporter ABCA1 moves lipids onto brain apoE and that lipidated apoE affects Ab and amyloid metabolism. In AD mice, ABCA1 deficiency exacerbates amyloidogenesis, whereas selective overexpression of ABCA1 ameliorates amyloid burden. Liver X Receptor (LXR) agonists such as GW3965, which stimulate ABCA1 and apoE expression, reduce Ab levels and rescue cognitive deficits in AD mice. However, LXR agonists induce a wide array of targets and the key genes that mediate their positive effects for AD are unknown. Methods: Here we tested the hypothesis that ABCA1 is required for the beneficial effects of GW3965 in the APP/PS1 model of AD, with and without ABCA1, using low-dose prophylactic, low-dose therapeutic, and high-dose therapeutic treatment arms. Mice in the prophylactic group received GW3965 in chow at 2.5 mg/kg/d from 1640 weeks of age. Mice in the low-dose therapeutic group received GW3965 in chow at 2.5 mg/kg/d from 32-40 week of age. Mice in the high-dose therapeutic group received GW3965 in chow at 33 mg/kg/ d from 32-40 week of age. All animals were analysed at 40 weeks of age. Results: Cognitive performance in the Novel Object Recognition task was restored to baseline in all three treatment arms with functional ABCA1, whereas performance remained impaired in treated mice lacking ABCA1. Importantly, cognitive improvement could be observed without significant changes in Ab levels, amyloid burden or apoE-HDL levels, suggesting that ABCA1 affects an Ab pool particularly important for cognitive function. Significant reductions in Ab and amyloid levels were observed only upon high-dose treatment, which provided no additional cognitive benefits compared to low-dose-treatment. Also, prophylactic treatment provided no additional benefits compared to therapeutic treatment, suggesting that LXR agonists may be effective for symptomatic AD patients. Conclusions: Our results suggest that ABCA1-mediated lipidation of apoE mediates many of the beneficial effects of LXR agonists on cognition and Ab metabolism and highlights ABCA1 as a potential AD therapeutic target. P1-259
ALZHEIMER‘S DISEASE AND GENE REGULATION: A POSSIBLE ROLE OF Aß IN THE NUCLEUS
Christian Barucker1, Anja Harmeier1, Joerg Weiske2, Kai Albring2, Otmar Huber2, Gerd Multhaup1, 1Free University Berlin, Berlin, Germany; 2 Friedrich-Schiller-University Jena, Jena, Germany. Contact e-mail: [email protected] Background: The pathogenic effects observed in AD are ascribed to soluble low-n oligomers of Aß42, although the exact mechanism is still unclear. Recently, we could show that cellular toxicity and LTP inhibition of Aß is not a simple cause of oligomerization but a consequence of a specific Aß-conformation determined by the GxxxG interaction motif. Aß42 peptides with an alanine substitution of glycine (G33A) were much less toxic than Aß42wt. Our findings unraveled that G33 is the key amino acid determining cellular activities of Aß. In the present study we have discovered a novel role for Aß42 in nuclear signaling, which is different from AICD. The observed involvement of the peptide in gene regulation was specific for Aß42 and could either represent a normal or a gain-of-function. Thus, we speculate that the major deleterious effects of Aß42 in the pathogenesis could be mediated through changing the activities of transcription factors and/ or altering the expression profiles of disease-modifying genes Methods: Neuroblastoma cells (SH-SY5Y) were treated with freshly dissolved synthetic Aß peptides. The internalization of these peptides was monitored by Western blot, ELISA and LSM. Using ChIP technique we could demonstrate that Aß specifically binds either directly or indirectly to promoters. To verify the functional binding of Aß peptides to chromatin we investigated the mRNA levels by qRTPCR in addition to the analysis of chromatin-bound protein complexes. Results: Different forms of Aß were rapidly internalized and transported within the cell from the cytoplasm into the nucleus of SH-SY5Y cells. There, Aß peptides accumulated in a time dependent manner as shown by three different methods. Only the accumulation of Aß42 led to changes in the expression of various mRNAs. Whereas the wild-type sequence affected mRNA
Poster Presentations P1
as the Ab42/Ab40 ratio, were increased in Purkinje neurons from sporadic AD cases as compared to controls. However, the levels of Ab42 as well as Ab40 were clearly lower in Purkinje neurons than in pyramidal neurons. Based on the volume of the captured neurons the intraneuronal molar concentrations of Ab were calculated. Conclusions: We suggest that the concentration of intracellular Ab42 correlates to the neuropathology of Alzheimer’s disease. P1-257
IMPACT OF CHOLESTEROL ON APP DIMERS’ FORMATION, USING FRET DETECTION AND FLUORESCENCE DECAY IMAGING
Viviane Devauges1,2, Catherine Marquer3, Ge´raldine Liot4, Pierre Blandin1,2, Jack-Christophe Cossec3, Sandrine Le´cart5, Sandrine Humbert4, Fre´de´ric Saudou4, Fre´de´ric Druon2,5, Patrick Georges2,5, Marie-Claude Potier3, Sandrine Le´veˆque-Fort1,5, 1Laboratoire de Photophysique Mole´culaire, Orsay, France; 2Laboratoire Charles Fabry de l’Institut d’Optique, Palaiseau, France; 3CRICM/UPMC, Paris, France; 4Institut Curie, Orsay, France; 5Centre de Photonique Biome´dicale, Orsay, France. Contact e-mail: [email protected] Background: One of the major hallmarks of Alzheimer’s disease (AD) is the presence in the brain of senile plaques, mainly composed of Ab peptide. It results mainly from the sequential cleavage of APP (amyloid precursor protein) by the b-secretase BACE1. Though it seems clear that APP can dimerize, the sequence implied in the dimerization process and the impact of APP dimerization on Ab secretion is still under debate. Recent genetic, epidemiologic and biochemical data point to a link between cholesterol and AD. We previously showed that increasing plasma membrane cholesterol content leads to a gain in APP-BACE1 proximity at the plasma membrane, increased APP endocytosis and a higher Ab production. We thus wondered how increasing membrane cholesterol would impact on APP dimerization. Methods: Two different FRET methods were developped to assess APP dimerization in living cells. Firstly, proximity (<10 nm) of APP-GFP and APP-mCherry was monitored by FRET-FLIM (Fluorescence Lifetime Imaging to monitor GFP’s lifetime). Then, homo-FRET between GFPs was followed using time-resolved fluorescence anisotropy imaging of cells expressing APP-GFP. These experiments were performed under both TIRF (plasma membrane only) and epifluorescence (whole cell) illumination. Modulation of membrane cholesterol was performed in HEK-293 cell lines and embryonic rat hippocampal neurons using a MbCD-cholesterol complex and was kinetically followed over 30 minutes. Results: First measurements were made with cytosolic GFP or with a membrane anchored GFPfusion protein to ensure that increasing cholesterol concentration at the plasma membrane of cells does not have an impact on GFP anisotropy on its own. Thus the effects observed on APP-GFP when adding cholesterol are specifically due to homo-FRET. We used constituvely dimerizing mutant APP as a positive control. We are currently investigating the effect of cholesterol on APP dimerization using FRET-FLIM and homo-FRET on both cell types. Comparative results will be presented. Conclusions: We developed two novel complementary FRET techniques to probe APP dimerization in living cells. They were used to unravel the role of cholesterol on the formation of APP dimers in HEK-293 cells and in neurons, thus giving new insight on a possible link between APP dimerization and Ab production. P1-258
ABCA1 MEDIATES THE BENEFICIAL EFFECTS OF THE LIVER-X-RECEPTOR AGONIST GW3965 ON AMYLOID LOAD AND OBJECT RECOGNITION MEMORY IN APP/PS1 ALZHEIMER MICE
Cheryl L. Wellington1, James Donkin1, Sophie Stukas1, Veronica HirschReinshagen1, Dhananjay Namjoshi1, Anna Wilkinson1, Sharon May1, Jennifer Chan1, Jianjia Fan1, Jon Collins2, 1University of British Columbia, Vancouver, BC, Canada; 2GlaxoSmithKline, Research Triangle Park, NC, USA. Contact e-mail: [email protected] Background: Apolipoprotein E (apoE) is the major cholesterol carrier in the brain and the most validated genetic risk factor for Alzheimer’s Disease
(AD), the leading cause of dementia in the elderly characterized by Ab and amyloid deposits in brain tissue. We have shown that the cholesterol transporter ABCA1 moves lipids onto brain apoE and that lipidated apoE affects Ab and amyloid metabolism. In AD mice, ABCA1 deficiency exacerbates amyloidogenesis, whereas selective overexpression of ABCA1 ameliorates amyloid burden. Liver X Receptor (LXR) agonists such as GW3965, which stimulate ABCA1 and apoE expression, reduce Ab levels and rescue cognitive deficits in AD mice. However, LXR agonists induce a wide array of targets and the key genes that mediate their positive effects for AD are unknown. Methods: Here we tested the hypothesis that ABCA1 is required for the beneficial effects of GW3965 in the APP/PS1 model of AD, with and without ABCA1, using low-dose prophylactic, low-dose therapeutic, and high-dose therapeutic treatment arms. Mice in the prophylactic group received GW3965 in chow at 2.5 mg/kg/d from 1640 weeks of age. Mice in the low-dose therapeutic group received GW3965 in chow at 2.5 mg/kg/d from 32-40 week of age. Mice in the high-dose therapeutic group received GW3965 in chow at 33 mg/kg/ d from 32-40 week of age. All animals were analysed at 40 weeks of age. Results: Cognitive performance in the Novel Object Recognition task was restored to baseline in all three treatment arms with functional ABCA1, whereas performance remained impaired in treated mice lacking ABCA1. Importantly, cognitive improvement could be observed without significant changes in Ab levels, amyloid burden or apoE-HDL levels, suggesting that ABCA1 affects an Ab pool particularly important for cognitive function. Significant reductions in Ab and amyloid levels were observed only upon high-dose treatment, which provided no additional cognitive benefits compared to low-dose-treatment. Also, prophylactic treatment provided no additional benefits compared to therapeutic treatment, suggesting that LXR agonists may be effective for symptomatic AD patients. Conclusions: Our results suggest that ABCA1-mediated lipidation of apoE mediates many of the beneficial effects of LXR agonists on cognition and Ab metabolism and highlights ABCA1 as a potential AD therapeutic target. P1-259
ALZHEIMER‘S DISEASE AND GENE REGULATION: A POSSIBLE ROLE OF Aß IN THE NUCLEUS
Christian Barucker1, Anja Harmeier1, Joerg Weiske2, Kai Albring2, Otmar Huber2, Gerd Multhaup1, 1Free University Berlin, Berlin, Germany; 2 Friedrich-Schiller-University Jena, Jena, Germany. Contact e-mail: [email protected] Background: The pathogenic effects observed in AD are ascribed to soluble low-n oligomers of Aß42, although the exact mechanism is still unclear. Recently, we could show that cellular toxicity and LTP inhibition of Aß is not a simple cause of oligomerization but a consequence of a specific Aß-conformation determined by the GxxxG interaction motif. Aß42 peptides with an alanine substitution of glycine (G33A) were much less toxic than Aß42wt. Our findings unraveled that G33 is the key amino acid determining cellular activities of Aß. In the present study we have discovered a novel role for Aß42 in nuclear signaling, which is different from AICD. The observed involvement of the peptide in gene regulation was specific for Aß42 and could either represent a normal or a gain-of-function. Thus, we speculate that the major deleterious effects of Aß42 in the pathogenesis could be mediated through changing the activities of transcription factors and/ or altering the expression profiles of disease-modifying genes Methods: Neuroblastoma cells (SH-SY5Y) were treated with freshly dissolved synthetic Aß peptides. The internalization of these peptides was monitored by Western blot, ELISA and LSM. Using ChIP technique we could demonstrate that Aß specifically binds either directly or indirectly to promoters. To verify the functional binding of Aß peptides to chromatin we investigated the mRNA levels by qRTPCR in addition to the analysis of chromatin-bound protein complexes. Results: Different forms of Aß were rapidly internalized and transported within the cell from the cytoplasm into the nucleus of SH-SY5Y cells. There, Aß peptides accumulated in a time dependent manner as shown by three different methods. Only the accumulation of Aß42 led to changes in the expression of various mRNAs. Whereas the wild-type sequence affected mRNA