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Gene regulation by gamma-secretase analyzed by microarray
Poster Presentations P1 P1-046
GENE REGULATION BY GAMMA-SECRETASE ANALYZED BY MICROARRAY
Sophie Restituito, Jiri Zavadil, Edward Ziff, Yutong Zhang, NYU Langone Medical Center, New York, New York. Background: Gamma-secretase, which generates amyloid peptide from APP, also cleaves other type I transmembrane proteins, including synaptic proteins such as N-cadherin. The cleavage pathway involves initial ectodomain shedding by a metalloprotease, followed by intramembrane cleavage by gamma-secretase. The C-terminal fragments generated from the cleavage of several of gamma-secretase substrates can have signaling functions and can regulate transcription (Hass et al., 2009)1. We have previously shown the existence of activity-dependent cleavage of APP and N-cadherin by metalloproteases and gamma-secretase at synapses. Moreover, these cleavages modify synaptic transmission and can thus provide a novel form of functional synaptic auto regulation. Methods: We thus assayed if gamma-secretase cleavage could modify gene regulation in neurons. We performed a microarray expression profiling analysis using RNA isolated from 14 DIV primary hippocampal neurons in culture treated with the gamma-secretase inhibitors, L685458 for 2h or 4h and compared them to untreated controls. The experiment was carried out in duplicate and the normalized array data were analyzed with the DAVID bioinformatic database and the Gene Set Enrichment Analysis (GSEA) tool. Results: Using the DAVID bioinformatic database, we observed a down regulation of genes involved in Notch receptor signaling, including HES1, as expected. Moreover, we found that apoptosis and Wnt signaling programs were remodeled based on both up regulated and down regulated genes. Interestingly a number of genes coding for potassium channels, adhesion molecules and synaptic proteins were down regulated. Analogously, using GSEA, we identified down regulation of genes involved in the Notch receptor signaling, as well as others involved in cell-cell adhesion, such as E-cadherin, and in synaptic transmission such as the NMDA NR2 receptor. Interestingly we also observed a down regulation of MT5MMP gene, a metalloprotease involved in N-cadherin cleavage in neurons, and of Presenilin1, one of the components of gamma-secretase. Conclusions: In conclusion, we generated a catalogue of candidate pathways involved in synaptic function that may be regulated by gamma-secretase. Reference: [1] Hass MR, Sato C, Kopan R, Zhao G (2009) Presenilin:RIP and beyond. Seminars in Cell & Developmental Biology 20:201-210. P1-047
MEMANTINE REVERSES COGNITIVE DEFICITS INDUCED BY TOTAL SLEEP DEPRIVATION IN O. DEGUS
Francisco Ros-Bernal1, Cesar Lucas2, Carmen Ros3, Virginia Izura4, Fabienne Aujard5, Yves Lamberty6, Elaine Irving7, Maria Herrero8, 1 NiCE-CIBERNED. University of Murcia, Murcia; 2NiCE-Ciberned. University of Murcia, Murcia; 3NiCE- Ciberned. University of Murcia, Murcia; 4NiCe-Ciberned. University of Murcia, Murcia; 5Centre National de la Recherche Scientifique UMR 7179, Brunoy; 6UCB Pharma, Brussels; 7 GlaxoSmithKline R&D, Stevenage; 8Director NiCe-Ciberned, University of Murcia, Murcia. Background: To date, preclinical models used to assess novel cognitive enhancing therapies have been poor predictors of positive clinical outcome. The Octodon degus expresses neuronal ß-amyloid precursor protein (displaying both intracellular and extracellular deposits of amyloid-ß-peptide), as well as intracellular accumulations of tau-protein and ubiquitin (Inestrosa et al., 2005). Within the frame of IMI PharmaCog European Consortium, we aimed to investigate the effect of sleep deprivation on O. degus cognitive performance to evaluate this as a potential model to assess the cognitive enhancing properties of novel therapeutic agents for Alzheimer’s Disease. Methods: A total of 20 Octodon degus (3 6 0.5 years) were randomly divided in 4 groups: control, Memantine (1-amino-3, 5-dimethyladamantane) treated and total sleep deprived with/without Memantine treatment. Total sleep deprivation (TSD) took place for six hours during three consecutive days. After each of these days we tested motor (by using an automated locomotor activity apparatus) and cognitive function (by using Barnes and Radial mazes) of all the animals. Memantine (10 mg/kg/day) or placebo was intraperitoneally administered before each sleep deprivation process.
S125
Results: All the sleep-deprived animals showed significant motor and cognitive deficits (p < 0.001) when compared with non-deprived (both control- and Memantine-treated) animals. However, Memantine significantly diminishes (p < 0.01) the time to complete the tasks and the number of errors in both radial and Barnes mazes in sleep deprived O. degus. Conclusions: These results indicate that systemically administered Memantine significantly counteracts the cognitive deficits induced by TSD in O. degus. Moreover, these results support the use of O. degus as a valuable model to perform TSD as a non pharmacological challenge to induce reversible cognitive deficits.
P1-048
CORRELATION OF COGNITIVE DEFICITS AND BRAIN CHOLINESTERASE ACTIVITY IN STREPTOZOTOCININTRACEREBROVENTRICULARLY TREATED RAT MODEL OF SPORADIC ALZHEIMER’S DISEASE
Melita Salkovic Petrisic1, Ana Knezovic2, Edna Gr€unblatt3, Martina K. Brueckner4, Thomas Arendt5, Siegfried Hoyer6, Peter Riederer7, 1 Medical School University of Zagreb, Zagreb; 2Medical School, University of Zagreb, Zagreb; 3University Z€urich-Irchel, Z€urich; 4University Leipzig, Paul Flechsig Institute of Brain Research, Leipzig; 5University Leipzig, Paul Flechsig Institute of Brain Research, Leipzig; 6University of Heidelberg, Heidelberg; 7Clinic for Psychiatry and Psychotherapy, Bayerische Julius-Maximilians-University of W€urzburg, Wuerzburg. Background: Cholinergic deficits in the brain play important role in the pathophysiology of cognitive deficits in sporadic Alzheimer’s disease (sAD). Rats treated intracerebroventricularly with streptozotocin (STZ-icv), have been proposed as an experimental sAD model. Increased brain cholinesterase activity and cognitive deficits in this model have been followed in literature up to 1 and 3 months after the STZ-icv injection, respectively. We investigated the effect of three STZ-icv doses on cognitive functions and brain cholinesterase activity in the STZ-icv rat model, three months after the STZ-icv administration. Methods: Adult Wistar rats were STZ-icv treated with 0.3, 1 and 3 mg/kg dose. Measurements of (1) cognitive deficits (Morris Water Maze/MWM/ and Passive Avoidance/PA/ tests), (2) cholinesterase activity in hippocampus (HPC) and parietotemporal cortex (PTC) (Elman’s method), (3) amyloid precursor protein mRNA (APP) expression (RT-PCR) and amyloid ß (Aß) accumulation (Aß immunohistochemistry), were explored 3 months after STZicv administration. Data were analyzed by Cruscal-Walles ANOVA and Mann-Whitney U test (P < 0.05). Results: STZ-icv (1 and 3 mg/kg) treated rats demonstrated memory retention deficits (PA: -46% and -66%, MWM: -36% and -38%, respectively, p < 0.05). STZ-icv 3 mg/kg dose caused a mild (+15%), insignificant (p ¼ 0.09) increase in HPC cholinesterase activity. Positive Aß signal was found in cerebral capillary walls of STZ-icv treated rats with no changes in APP mRNA production. Conclusions: Three months after STZ-icv administration, rat sAD model develops marked dosedependent cognitive deficits, which are followed by only mildly changed brain cholinesterase activity. In contrast to that, three months after ST-icv
GENE REGULATION BY GAMMA-SECRETASE ANALYZED BY MICROARRAY
Sophie Restituito, Jiri Zavadil, Edward Ziff, Yutong Zhang, NYU Langone Medical Center, New York, New York. Background: Gamma-secretase, which generates amyloid peptide from APP, also cleaves other type I transmembrane proteins, including synaptic proteins such as N-cadherin. The cleavage pathway involves initial ectodomain shedding by a metalloprotease, followed by intramembrane cleavage by gamma-secretase. The C-terminal fragments generated from the cleavage of several of gamma-secretase substrates can have signaling functions and can regulate transcription (Hass et al., 2009)1. We have previously shown the existence of activity-dependent cleavage of APP and N-cadherin by metalloproteases and gamma-secretase at synapses. Moreover, these cleavages modify synaptic transmission and can thus provide a novel form of functional synaptic auto regulation. Methods: We thus assayed if gamma-secretase cleavage could modify gene regulation in neurons. We performed a microarray expression profiling analysis using RNA isolated from 14 DIV primary hippocampal neurons in culture treated with the gamma-secretase inhibitors, L685458 for 2h or 4h and compared them to untreated controls. The experiment was carried out in duplicate and the normalized array data were analyzed with the DAVID bioinformatic database and the Gene Set Enrichment Analysis (GSEA) tool. Results: Using the DAVID bioinformatic database, we observed a down regulation of genes involved in Notch receptor signaling, including HES1, as expected. Moreover, we found that apoptosis and Wnt signaling programs were remodeled based on both up regulated and down regulated genes. Interestingly a number of genes coding for potassium channels, adhesion molecules and synaptic proteins were down regulated. Analogously, using GSEA, we identified down regulation of genes involved in the Notch receptor signaling, as well as others involved in cell-cell adhesion, such as E-cadherin, and in synaptic transmission such as the NMDA NR2 receptor. Interestingly we also observed a down regulation of MT5MMP gene, a metalloprotease involved in N-cadherin cleavage in neurons, and of Presenilin1, one of the components of gamma-secretase. Conclusions: In conclusion, we generated a catalogue of candidate pathways involved in synaptic function that may be regulated by gamma-secretase. Reference: [1] Hass MR, Sato C, Kopan R, Zhao G (2009) Presenilin:RIP and beyond. Seminars in Cell & Developmental Biology 20:201-210. P1-047
MEMANTINE REVERSES COGNITIVE DEFICITS INDUCED BY TOTAL SLEEP DEPRIVATION IN O. DEGUS
Francisco Ros-Bernal1, Cesar Lucas2, Carmen Ros3, Virginia Izura4, Fabienne Aujard5, Yves Lamberty6, Elaine Irving7, Maria Herrero8, 1 NiCE-CIBERNED. University of Murcia, Murcia; 2NiCE-Ciberned. University of Murcia, Murcia; 3NiCE- Ciberned. University of Murcia, Murcia; 4NiCe-Ciberned. University of Murcia, Murcia; 5Centre National de la Recherche Scientifique UMR 7179, Brunoy; 6UCB Pharma, Brussels; 7 GlaxoSmithKline R&D, Stevenage; 8Director NiCe-Ciberned, University of Murcia, Murcia. Background: To date, preclinical models used to assess novel cognitive enhancing therapies have been poor predictors of positive clinical outcome. The Octodon degus expresses neuronal ß-amyloid precursor protein (displaying both intracellular and extracellular deposits of amyloid-ß-peptide), as well as intracellular accumulations of tau-protein and ubiquitin (Inestrosa et al., 2005). Within the frame of IMI PharmaCog European Consortium, we aimed to investigate the effect of sleep deprivation on O. degus cognitive performance to evaluate this as a potential model to assess the cognitive enhancing properties of novel therapeutic agents for Alzheimer’s Disease. Methods: A total of 20 Octodon degus (3 6 0.5 years) were randomly divided in 4 groups: control, Memantine (1-amino-3, 5-dimethyladamantane) treated and total sleep deprived with/without Memantine treatment. Total sleep deprivation (TSD) took place for six hours during three consecutive days. After each of these days we tested motor (by using an automated locomotor activity apparatus) and cognitive function (by using Barnes and Radial mazes) of all the animals. Memantine (10 mg/kg/day) or placebo was intraperitoneally administered before each sleep deprivation process.
S125
Results: All the sleep-deprived animals showed significant motor and cognitive deficits (p < 0.001) when compared with non-deprived (both control- and Memantine-treated) animals. However, Memantine significantly diminishes (p < 0.01) the time to complete the tasks and the number of errors in both radial and Barnes mazes in sleep deprived O. degus. Conclusions: These results indicate that systemically administered Memantine significantly counteracts the cognitive deficits induced by TSD in O. degus. Moreover, these results support the use of O. degus as a valuable model to perform TSD as a non pharmacological challenge to induce reversible cognitive deficits.
P1-048
CORRELATION OF COGNITIVE DEFICITS AND BRAIN CHOLINESTERASE ACTIVITY IN STREPTOZOTOCININTRACEREBROVENTRICULARLY TREATED RAT MODEL OF SPORADIC ALZHEIMER’S DISEASE
Melita Salkovic Petrisic1, Ana Knezovic2, Edna Gr€unblatt3, Martina K. Brueckner4, Thomas Arendt5, Siegfried Hoyer6, Peter Riederer7, 1 Medical School University of Zagreb, Zagreb; 2Medical School, University of Zagreb, Zagreb; 3University Z€urich-Irchel, Z€urich; 4University Leipzig, Paul Flechsig Institute of Brain Research, Leipzig; 5University Leipzig, Paul Flechsig Institute of Brain Research, Leipzig; 6University of Heidelberg, Heidelberg; 7Clinic for Psychiatry and Psychotherapy, Bayerische Julius-Maximilians-University of W€urzburg, Wuerzburg. Background: Cholinergic deficits in the brain play important role in the pathophysiology of cognitive deficits in sporadic Alzheimer’s disease (sAD). Rats treated intracerebroventricularly with streptozotocin (STZ-icv), have been proposed as an experimental sAD model. Increased brain cholinesterase activity and cognitive deficits in this model have been followed in literature up to 1 and 3 months after the STZ-icv injection, respectively. We investigated the effect of three STZ-icv doses on cognitive functions and brain cholinesterase activity in the STZ-icv rat model, three months after the STZ-icv administration. Methods: Adult Wistar rats were STZ-icv treated with 0.3, 1 and 3 mg/kg dose. Measurements of (1) cognitive deficits (Morris Water Maze/MWM/ and Passive Avoidance/PA/ tests), (2) cholinesterase activity in hippocampus (HPC) and parietotemporal cortex (PTC) (Elman’s method), (3) amyloid precursor protein mRNA (APP) expression (RT-PCR) and amyloid ß (Aß) accumulation (Aß immunohistochemistry), were explored 3 months after STZicv administration. Data were analyzed by Cruscal-Walles ANOVA and Mann-Whitney U test (P < 0.05). Results: STZ-icv (1 and 3 mg/kg) treated rats demonstrated memory retention deficits (PA: -46% and -66%, MWM: -36% and -38%, respectively, p < 0.05). STZ-icv 3 mg/kg dose caused a mild (+15%), insignificant (p ¼ 0.09) increase in HPC cholinesterase activity. Positive Aß signal was found in cerebral capillary walls of STZ-icv treated rats with no changes in APP mRNA production. Conclusions: Three months after STZ-icv administration, rat sAD model develops marked dosedependent cognitive deficits, which are followed by only mildly changed brain cholinesterase activity. In contrast to that, three months after ST-icv