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Calcineurin inhibition alters APP metabolism in vitro and in vivo
Poster Presentations: P1 dementia. Conclusions: Our findings indicate that following cardiac surgery the number of individuals identified as at risk of dementia is much higher than expected in the general population (0.3-0.8% per year) as more than 50% of individuals were affected. Equally concerning are the results following non-cardiac surgery and minor procedures at one year showing 13% and 18% (respectively) of individuals at risk of dementia. These results may have significant implications for clinical decision making in healthcare, and may provide a model for therapeutic intervention AD trials. References: 1 Evered, L et al. Anesth Analg 2011; 112: 1179-85, 2 Xie,Z and Tanzie, R. Exp Gerontol 2006; 41:346-59, 3 Baranov, D et al. Anesth Analg 2009; 108:1627-30, 4 Silbert et al. Anesthesiology 2006; 104: 1137-45. P1-298
MEMANTINE: ASSESSMENT IN NOVEL OBJECT RECOGNITION AND QUANTITATIVE 2-DEOXYGLUCOSE METABOLISM IN RAT
Toshal Patel, Jonathan Papworth, Janette Robertson, Anthony Groom, Jane Gartlon, Adam Lucas, Alexander Syme, Lee Dawson, Eisai Ltd., Hatfield, United Kingdom. Background: The NMDA receptor antagonist memantine is currently one of the “Gold Standard” therapeutics used in treating dementia. The efficacy of memantine has been demonstrated in numerous cognition models (Mobius et al., 2004; Martinez-Coria et al., 2010); however, a detailed analysis of the neurocircuitry involved in cognitive enhancement has not been conducted. Methods: In the current study, we have compared the effect of memantine in a rat model of recognition memory, thought to involve perirhinal cortex-hippocampal circuit i.e. novel object recognition (NOR), with quantitative 2-deoxyglucose (2-DG) autoradiography to understand the underlying neurocircuitry. The use of these techniques could provide translation methodologies to understand the mechanism of such agents. Results: Memantine’s effects (3, 10 and 30 mg/kg, s.c.) was examined using 2-DG autoradiography, in a total of 37 brain regions, in conscious rats. Memantine induced significant (P<0.05) dose-dependent increases in glucose utilization in limbic areas; e.g., posterior cingulate cortex (11-40%), hippocampus (1-58%), anterior thalamus (6-40%). Reductions in glucose usage were observed in the auditory cortex (0-23%), medial geniculate (1-15%) and inferior colliculus (20-22%). In the rat NOR, memantine (0.3, 1 and 3mg/kg i.p.; ptt 1 h) did not improve object recognition at the doses examined. Bioanalysis from the NOR study revealed free brain concentrations of 0.18uM, 0.50uM and 1.73uM (0.3, 1 and 3 mg/kg, i.p., respectively). Bioanalysis from the 2-DG study revealed free brain concentrations of 1.8uM and 13.9uM (3 and 30mg/kg, s.c. respectively). While memantine did not enhance object discrimination at the doses examined, we were unable to examine higher doses due to induction of stereotype behaviour in animals treated with >10 mg/ kg. Conclusions: Memantine’s effects on glucose utilization are consistent with its activity as an NMDA receptor antagonist, similar previous reports for MK-801 (Kurumaji et al. 1989). While a direct correlation cannot be made between the effects in the NOR and 2-DG in the current studies, activation of glutamatergic circuitry in the hippocampus/ limbic system may be associated with the clinical pro-cognitive effects of this agent. However, the ability to use 2-DG as a truly translatable indicator for cognitive efficacy may suffer from sensitivity issues at least within the limited preclinical studies reported here. P1-299
CALCINEURIN INHIBITION ALTERS APP METABOLISM IN VITRO AND IN VIVO
Aleister Saunders, Suruchi Utreja, Brie Paddock, Sean Miller, Daniel R. Marenda, Drexel University, Philadelphia, Pennsylvania, United States.
P209
Background: APP metabolism plays a central role in Alzheimer’s disease (AD) pathogenesis. APP metabolism is complex and includes gene/protein expression, trafficking, post-translational modification, and regulated intramembraneous proteolysis. To identify these genes we carried out human cell-based genetic screen for APP metabolism regulators, by individually knocking-down human genes located in regions of the human genome implicated by genetic studies. Methods: Bioinformatic tools were used to identify all genes in regions of Chr. 9 and Chr. 10 genetically linked to AD. Calcineurin (CaN) expression was knocked-down or over-expressed in human cell lines. FK506 was used to inhibit CaN activity in cells and in a transgenic Drosophila AD models. Western blot and ELISA analysis of human cell lines and Drosophila brains was used to assess alteration in APP metabolism. The effect of CaN inhibition on Drosophila complex motor function, learning and memory was also investigated. Results: We demonstrate that knock-down and over-expression of CaN catalytic (PPP3CB) and regulatory (PPP3R2) subunits alters APP metabolism in human cell lines. FK506 mediated inhibition of CaN activity resulted in similar effects as catalytic subunit knock-down. Using two Drosophila AD models, we demonstrate that CaN inhibition alters APP processing, reduces Abeta puncta in Drosophila brain, reduces complex motor function deficits, and reduces memory deficits. Conclusions: This work demonstrates that CaN regulates APP metabolism in vitro and in vivo. Inhibition of calcineurin with FDA approved drugs results in reduction of AD related phenotypes in vivo. This suggests that targeting CaN may have clinical relevance in the treatment of AD.
P1-300
SUPPRESSION OF NFKB SIGNALING PATHWAY BY EPIGALLOCATECHIN GALLATE ATTENUATES DIABETIC ENCEPHALOPATHY
Anurag Kuhad, Kanwaljit Chopra, Panjab University, Chandigarh, India. Background: Diabetes mellitus produces numerous neurophysiological and structural changes in the brain and it is associated with moderate cognitive deficits. The etiology of diabetes associated cognitive decline is multifactorial and involves insulin receptor down regulation, neuronal apoptosis and glutamatergic neurotransmission. The study was designed to evaluate the impact of epigallocatechin gallate on cognitive function and neuroinflammatory cascade in streptozotocin-induced diabetes. Methods: Streptozotocin-induced diabetic rats were treated with epigallocatechin gallate or with vehicle for 10 weeks. Morris water maze was used for behavioral assessment of memory. Cytoplasmic and nuclear fractions of cerebral cortex and hippocampus were prepared for the quantification of acetylcholinestrease activity, oxidative-nitrosative stress (lipid peroxidation, superoxide dismutase, catalase, non protein thiols, total nitric oxide), tumor necrosis factor-alpha (TNF-a), interleukin-1beta (IL-1b), p56 subunit of NFKb and caspase-3. Results: After 10 weeks of steptozotocin injection, the rats produced significant increase in transfer latency which was coupled with enhanced acetylcholinestrease activity, increased oxidative-nitrosative stress, TNF-a, IL-1b, caspase-3 activity in cytoplasmic lysate and active p65 subunit of NFKb in nuclear lysate of cerebral cortex and hippocampus regions of diabetic rat brain. Interestingly, co-administration of epigallocatechin gallate significantly and dosedependently prevented behavioral, biochemical and molecular changes associated with diabetes. Moreover, diabetic rats treated with insulinepigallocatechin gallate combination produced more pronounced effect on molecular parameters as compared to their per se groups. Conclusions: Collectively, the data reveal that activation of NFKb signaling pathway is associated with diabetes induced cognitive impairment and point towards the therapeutic potential of epigallocatechin gallate in diabetic encephalopathy.
MEMANTINE: ASSESSMENT IN NOVEL OBJECT RECOGNITION AND QUANTITATIVE 2-DEOXYGLUCOSE METABOLISM IN RAT
Toshal Patel, Jonathan Papworth, Janette Robertson, Anthony Groom, Jane Gartlon, Adam Lucas, Alexander Syme, Lee Dawson, Eisai Ltd., Hatfield, United Kingdom. Background: The NMDA receptor antagonist memantine is currently one of the “Gold Standard” therapeutics used in treating dementia. The efficacy of memantine has been demonstrated in numerous cognition models (Mobius et al., 2004; Martinez-Coria et al., 2010); however, a detailed analysis of the neurocircuitry involved in cognitive enhancement has not been conducted. Methods: In the current study, we have compared the effect of memantine in a rat model of recognition memory, thought to involve perirhinal cortex-hippocampal circuit i.e. novel object recognition (NOR), with quantitative 2-deoxyglucose (2-DG) autoradiography to understand the underlying neurocircuitry. The use of these techniques could provide translation methodologies to understand the mechanism of such agents. Results: Memantine’s effects (3, 10 and 30 mg/kg, s.c.) was examined using 2-DG autoradiography, in a total of 37 brain regions, in conscious rats. Memantine induced significant (P<0.05) dose-dependent increases in glucose utilization in limbic areas; e.g., posterior cingulate cortex (11-40%), hippocampus (1-58%), anterior thalamus (6-40%). Reductions in glucose usage were observed in the auditory cortex (0-23%), medial geniculate (1-15%) and inferior colliculus (20-22%). In the rat NOR, memantine (0.3, 1 and 3mg/kg i.p.; ptt 1 h) did not improve object recognition at the doses examined. Bioanalysis from the NOR study revealed free brain concentrations of 0.18uM, 0.50uM and 1.73uM (0.3, 1 and 3 mg/kg, i.p., respectively). Bioanalysis from the 2-DG study revealed free brain concentrations of 1.8uM and 13.9uM (3 and 30mg/kg, s.c. respectively). While memantine did not enhance object discrimination at the doses examined, we were unable to examine higher doses due to induction of stereotype behaviour in animals treated with >10 mg/ kg. Conclusions: Memantine’s effects on glucose utilization are consistent with its activity as an NMDA receptor antagonist, similar previous reports for MK-801 (Kurumaji et al. 1989). While a direct correlation cannot be made between the effects in the NOR and 2-DG in the current studies, activation of glutamatergic circuitry in the hippocampus/ limbic system may be associated with the clinical pro-cognitive effects of this agent. However, the ability to use 2-DG as a truly translatable indicator for cognitive efficacy may suffer from sensitivity issues at least within the limited preclinical studies reported here. P1-299
CALCINEURIN INHIBITION ALTERS APP METABOLISM IN VITRO AND IN VIVO
Aleister Saunders, Suruchi Utreja, Brie Paddock, Sean Miller, Daniel R. Marenda, Drexel University, Philadelphia, Pennsylvania, United States.
P209
Background: APP metabolism plays a central role in Alzheimer’s disease (AD) pathogenesis. APP metabolism is complex and includes gene/protein expression, trafficking, post-translational modification, and regulated intramembraneous proteolysis. To identify these genes we carried out human cell-based genetic screen for APP metabolism regulators, by individually knocking-down human genes located in regions of the human genome implicated by genetic studies. Methods: Bioinformatic tools were used to identify all genes in regions of Chr. 9 and Chr. 10 genetically linked to AD. Calcineurin (CaN) expression was knocked-down or over-expressed in human cell lines. FK506 was used to inhibit CaN activity in cells and in a transgenic Drosophila AD models. Western blot and ELISA analysis of human cell lines and Drosophila brains was used to assess alteration in APP metabolism. The effect of CaN inhibition on Drosophila complex motor function, learning and memory was also investigated. Results: We demonstrate that knock-down and over-expression of CaN catalytic (PPP3CB) and regulatory (PPP3R2) subunits alters APP metabolism in human cell lines. FK506 mediated inhibition of CaN activity resulted in similar effects as catalytic subunit knock-down. Using two Drosophila AD models, we demonstrate that CaN inhibition alters APP processing, reduces Abeta puncta in Drosophila brain, reduces complex motor function deficits, and reduces memory deficits. Conclusions: This work demonstrates that CaN regulates APP metabolism in vitro and in vivo. Inhibition of calcineurin with FDA approved drugs results in reduction of AD related phenotypes in vivo. This suggests that targeting CaN may have clinical relevance in the treatment of AD.
P1-300
SUPPRESSION OF NFKB SIGNALING PATHWAY BY EPIGALLOCATECHIN GALLATE ATTENUATES DIABETIC ENCEPHALOPATHY
Anurag Kuhad, Kanwaljit Chopra, Panjab University, Chandigarh, India. Background: Diabetes mellitus produces numerous neurophysiological and structural changes in the brain and it is associated with moderate cognitive deficits. The etiology of diabetes associated cognitive decline is multifactorial and involves insulin receptor down regulation, neuronal apoptosis and glutamatergic neurotransmission. The study was designed to evaluate the impact of epigallocatechin gallate on cognitive function and neuroinflammatory cascade in streptozotocin-induced diabetes. Methods: Streptozotocin-induced diabetic rats were treated with epigallocatechin gallate or with vehicle for 10 weeks. Morris water maze was used for behavioral assessment of memory. Cytoplasmic and nuclear fractions of cerebral cortex and hippocampus were prepared for the quantification of acetylcholinestrease activity, oxidative-nitrosative stress (lipid peroxidation, superoxide dismutase, catalase, non protein thiols, total nitric oxide), tumor necrosis factor-alpha (TNF-a), interleukin-1beta (IL-1b), p56 subunit of NFKb and caspase-3. Results: After 10 weeks of steptozotocin injection, the rats produced significant increase in transfer latency which was coupled with enhanced acetylcholinestrease activity, increased oxidative-nitrosative stress, TNF-a, IL-1b, caspase-3 activity in cytoplasmic lysate and active p65 subunit of NFKb in nuclear lysate of cerebral cortex and hippocampus regions of diabetic rat brain. Interestingly, co-administration of epigallocatechin gallate significantly and dosedependently prevented behavioral, biochemical and molecular changes associated with diabetes. Moreover, diabetic rats treated with insulinepigallocatechin gallate combination produced more pronounced effect on molecular parameters as compared to their per se groups. Conclusions: Collectively, the data reveal that activation of NFKb signaling pathway is associated with diabetes induced cognitive impairment and point towards the therapeutic potential of epigallocatechin gallate in diabetic encephalopathy.