- Email: [email protected]
P1-458: Effect of disease-associated mutations in the prion protein on the β-secretase cleavage of the amyloid precursor protein
T354
Poster Presentations P1
against different neuronal proteins, as well as time-lapse movies using a CellR/Olympus microscope and electrophysiological recordings using Axioclamp 10.2 software. Results: Both WT and tKO ES cells gave rise to highly pure neuronal cultures. They differentiated in a similar way through a stage of Pax6⫹ radial glia cells. No migration defect was observed on a laminin surface. Neuronal polarity was established in both groups with segregation of a Tau⫹ axon and several Map2⫹ dendrites. The main phenotype observed were multipolar neurons. The tKO neurons also formed long axons. At 2 weeks, Synaptophysin ⫹ synaptic contacts were established. Like the WT cells, the APP tKO cells gave rise to mainly glutamatergic neurons. After 2 weeks in culture spontaneous synaptic currents could be recorded from both WT and tKO neurons. Conclusions: To try to unravel the function of APP we differentiated APP/APLP1/ APLP2 triple KO ES cells into glutamatergic neurons. Our results indicate that despite the absence of APP these neurons derived from tKO ES cells can differentiate, establish neuronal polarity and form active synaptic contacts. Acknowledgements: We would like to thank Ulrike Mu¨ller for kindly providing the mice from which we derived the tKO ES cells and Miriam Bibel for providing us with a WT ES cell line. B.A.B. is a Research Assistant of the Fund for Scientific Research Flanders. P1-458
EFFECT OF DISEASE-ASSOCIATED MUTATIONS IN THE PRION PROTEIN ON THE -SECRETASE CLEAVAGE OF THE AMYLOID PRECURSOR PROTEIN
Isobel J. Morten1, Heledd H. Griffiths1, Herbert N. Baybutt2, Jean Manson2, Roberto Chiesa3, Nigel M. Hooper1, 1University of Leeds, Leeds, United Kingdom; 2Roslin Institute, Edinburgh, United Kingdom; 3Dulbecco Telethon Institute, Milano, Italy. Contact e-mail: [email protected] Background: The cellular form of the prion protein (PrPC), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Scheinker-Straussler (GSS) disease in humans, has been shown to inhibit the initial step of the proteolytic processing of the amyloid precursor protein (APP). PrPC inhibits the -site APP cleaving enzyme (BACE1) resulting in the reduction of the amyloid  (A) peptides, which are involved in the pathogenesis of Alzheimer’s disease (AD). In SH-SY5Y cells, PrPC expression reduced secretion into the conditioned medium of A40 by 92% and A42 to undetectable levels, and reduced sAPP shedding by 98%. Studies using PrP null mice showed a significant increase in A40 and A42 in the brain, providing direct evidence that PrPC regulates the production of A in vivo. Two mutants of PrP, PG14 and A116V, which are associated with CJD and GSS, respectively, did not inhibit the -cleavage of APP when expressed in SH-SY5Y cells. Here we have examined the effect of other diseaseassociated mutations in PrPC on the BACE1 cleavage of APP. Methods: SH-SY5Y cells expressing APP and various mutant PrP constructs associated with human prion diseases were established. Cell lysates and conditioned medium were subjected to western blot using antibodies specific to PrP, APP, sAPP, sAPP␣ and actin, and BACE1 activity was assayed using a quenched fluorescent peptide substrate. Results: Two mutants of murine PrPC (W144stop and Q159stop equivalent to Y145stop and Q160stop in human PrPC that are associated with familial prion diseases) were rapidly metabolised by the proteasome and failed to inhibit the BACE1 cleavage of APP. The effect of further mutants of PrPC, including P101L and D177N, on the BACE1 cleavage of APP is currently being examined. In addition, the levels of A peptides in the brains from transgenic mice expressing some of these mutants will be reported. Conclusions: The mechanism by which PrPC inhibits A peptide production and the lack of this regulatory control by mutants of PrPC associated with prion diseases suggests that an increase of A peptides could contribute to disease pathogenesis. These observations have implications for both prion disease research and AD.
P1-459
ENDOTHELIN-CONVERTING ENZYME-1 AND -2 LEVELS ARE INCREASED IN ALZHEIMER’S DISEASE
Jennifer C. Palmer, Seth Love, Patrick G. Kehoe, University of Bristol, Bristol, United Kingdom. Contact e-mail: [email protected] Background: Accumulation of amyloid beta (A) within the brain is thought to be central to the pathogenesis of Alzheimer’s disease (AD). An increase in A production has been shown in familial AD but it remains unclear whether production is increased in the more common sporadic AD (SAD). Accumulation of A in SAD may be due to decreased clearance, one mechanism of which is enzyme-mediated catabolism. Endothelinconverting enzymes-1 and -2 (ECE-1 and -2) are capable of degrading A in vitro and in vivo and are both expressed in the human brain. However, apart from a single report of decreased ECE-2 mRNA, there is little published data on the expression of either of these enzymes in AD. Objectives: Our aim in this study was to compare ECE-2 protein and ECE-1 and -2 mRNA levels between AD and control brains. Methods: We analysed homogenates of temporal neocortex from 15 neuropathologicallyconfirmed cases of SAD and 15 paired controls, matched for age at death (to within 5 years) and post-mortem delay (to within 5 hours). The age range was 57-93 years (mean 77.3) and post-mortem delay 4-42 hours (mean 21.3) in the AD cases, and 58-93 years (mean 78) and 3-36 hours (mean 21.3) in the controls. mRNA levels were measured by RT-PCR on triplicate samples, and gene expression relative to GAPDH, smooth muscle actin and neuron-specific enolase was calculated by the 2-⌬⌬Ct method. Protein levels were measured by sandwich ELISA, using antibodies that we validated by western blot. Results: ECE-1 and -2 mRNA levels were significantly higher in AD than control brains. Likewise, ECE-2 protein levels were significantly elevated in AD. Conclusions: We have shown significant increases in the expression of both ECE-1 and -2 in AD brain. Our findings suggest that accumulation of A in SAD is not caused by reduced levels of ECE-1 and -2. The increased expression of these enzymes in AD may be a response to increases in A. P1-460
MATRIX METALLOPROTEINASE INHIBITION REDUCES OXIDATIVE STRESS ASSOCIATED WITH CEREBRAL AMYLOID ANGIOPATHY IN VIVO IN TRANSGENIC MICE
Monica Garcia-Alloza, Claudia Prada, Elissa M. Robbins, Rebecca A. Betensky, Steven M. Greenberg, Matthew P. Frosch, Brian J. Bacskai, Massachusetts General Hospital, Charlestown Navy Yard, MA, USA. Contact e-mail: [email protected] Background: Cerebral amyloid angiopathy (CAA), characterized by extracellular -amyloid peptide (A) deposits in the vessel walls, is present in the majority of cases of Alzheimer’s disease and is a major cause of hemorrhagic stroke. The molecular pathways activated by vascular A in causing breakdown of the vessel wall are poorly understood. Among potential candidates are A-induced synthesis, release and activation of the extracellular matrix metalloproteinases (MMP) and A-induced oxidative stress. Methods: APPswe/PS1dE9 mice (6-7 months old) and tg-2576 mice (12.5-14 months old) had permanent cranial windows placed. We used in vivo multiphoton microscopy to measure: 1) progression of CAA using methoxy-XO4, 2) activation of MMP with a green fluorescent substrate (DQ gelatin), and 3) accumulation of reactive oxygen species (ROS) with the fluorescent probe Amplex Red. Quantitative analysis of CAA burden, Amplex Red signal and MMP signal was performed using Image J and Adobe Photoshop 7. Results: Using this system, we observed that MMP activation and oxidative stress were both associated with vessel segments affected with CAA. Antioxidant treatment with N-tert-butyl-␣phenil-nitrone (PBN) was capable of reducing oxidative stress associated with CAA (⬃50% reduction, *P⬍0.001 vs. Control) without affecting MMP activation. Conversely, a selection of MMP inhibitors with different mechanisms of action, including minocycline, simvastatin and GM6001, reduced not only CAA-associated MMP activation (⬃30-40% reduction,
Poster Presentations P1
against different neuronal proteins, as well as time-lapse movies using a CellR/Olympus microscope and electrophysiological recordings using Axioclamp 10.2 software. Results: Both WT and tKO ES cells gave rise to highly pure neuronal cultures. They differentiated in a similar way through a stage of Pax6⫹ radial glia cells. No migration defect was observed on a laminin surface. Neuronal polarity was established in both groups with segregation of a Tau⫹ axon and several Map2⫹ dendrites. The main phenotype observed were multipolar neurons. The tKO neurons also formed long axons. At 2 weeks, Synaptophysin ⫹ synaptic contacts were established. Like the WT cells, the APP tKO cells gave rise to mainly glutamatergic neurons. After 2 weeks in culture spontaneous synaptic currents could be recorded from both WT and tKO neurons. Conclusions: To try to unravel the function of APP we differentiated APP/APLP1/ APLP2 triple KO ES cells into glutamatergic neurons. Our results indicate that despite the absence of APP these neurons derived from tKO ES cells can differentiate, establish neuronal polarity and form active synaptic contacts. Acknowledgements: We would like to thank Ulrike Mu¨ller for kindly providing the mice from which we derived the tKO ES cells and Miriam Bibel for providing us with a WT ES cell line. B.A.B. is a Research Assistant of the Fund for Scientific Research Flanders. P1-458
EFFECT OF DISEASE-ASSOCIATED MUTATIONS IN THE PRION PROTEIN ON THE -SECRETASE CLEAVAGE OF THE AMYLOID PRECURSOR PROTEIN
Isobel J. Morten1, Heledd H. Griffiths1, Herbert N. Baybutt2, Jean Manson2, Roberto Chiesa3, Nigel M. Hooper1, 1University of Leeds, Leeds, United Kingdom; 2Roslin Institute, Edinburgh, United Kingdom; 3Dulbecco Telethon Institute, Milano, Italy. Contact e-mail: [email protected] Background: The cellular form of the prion protein (PrPC), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Scheinker-Straussler (GSS) disease in humans, has been shown to inhibit the initial step of the proteolytic processing of the amyloid precursor protein (APP). PrPC inhibits the -site APP cleaving enzyme (BACE1) resulting in the reduction of the amyloid  (A) peptides, which are involved in the pathogenesis of Alzheimer’s disease (AD). In SH-SY5Y cells, PrPC expression reduced secretion into the conditioned medium of A40 by 92% and A42 to undetectable levels, and reduced sAPP shedding by 98%. Studies using PrP null mice showed a significant increase in A40 and A42 in the brain, providing direct evidence that PrPC regulates the production of A in vivo. Two mutants of PrP, PG14 and A116V, which are associated with CJD and GSS, respectively, did not inhibit the -cleavage of APP when expressed in SH-SY5Y cells. Here we have examined the effect of other diseaseassociated mutations in PrPC on the BACE1 cleavage of APP. Methods: SH-SY5Y cells expressing APP and various mutant PrP constructs associated with human prion diseases were established. Cell lysates and conditioned medium were subjected to western blot using antibodies specific to PrP, APP, sAPP, sAPP␣ and actin, and BACE1 activity was assayed using a quenched fluorescent peptide substrate. Results: Two mutants of murine PrPC (W144stop and Q159stop equivalent to Y145stop and Q160stop in human PrPC that are associated with familial prion diseases) were rapidly metabolised by the proteasome and failed to inhibit the BACE1 cleavage of APP. The effect of further mutants of PrPC, including P101L and D177N, on the BACE1 cleavage of APP is currently being examined. In addition, the levels of A peptides in the brains from transgenic mice expressing some of these mutants will be reported. Conclusions: The mechanism by which PrPC inhibits A peptide production and the lack of this regulatory control by mutants of PrPC associated with prion diseases suggests that an increase of A peptides could contribute to disease pathogenesis. These observations have implications for both prion disease research and AD.
P1-459
ENDOTHELIN-CONVERTING ENZYME-1 AND -2 LEVELS ARE INCREASED IN ALZHEIMER’S DISEASE
Jennifer C. Palmer, Seth Love, Patrick G. Kehoe, University of Bristol, Bristol, United Kingdom. Contact e-mail: [email protected] Background: Accumulation of amyloid beta (A) within the brain is thought to be central to the pathogenesis of Alzheimer’s disease (AD). An increase in A production has been shown in familial AD but it remains unclear whether production is increased in the more common sporadic AD (SAD). Accumulation of A in SAD may be due to decreased clearance, one mechanism of which is enzyme-mediated catabolism. Endothelinconverting enzymes-1 and -2 (ECE-1 and -2) are capable of degrading A in vitro and in vivo and are both expressed in the human brain. However, apart from a single report of decreased ECE-2 mRNA, there is little published data on the expression of either of these enzymes in AD. Objectives: Our aim in this study was to compare ECE-2 protein and ECE-1 and -2 mRNA levels between AD and control brains. Methods: We analysed homogenates of temporal neocortex from 15 neuropathologicallyconfirmed cases of SAD and 15 paired controls, matched for age at death (to within 5 years) and post-mortem delay (to within 5 hours). The age range was 57-93 years (mean 77.3) and post-mortem delay 4-42 hours (mean 21.3) in the AD cases, and 58-93 years (mean 78) and 3-36 hours (mean 21.3) in the controls. mRNA levels were measured by RT-PCR on triplicate samples, and gene expression relative to GAPDH, smooth muscle actin and neuron-specific enolase was calculated by the 2-⌬⌬Ct method. Protein levels were measured by sandwich ELISA, using antibodies that we validated by western blot. Results: ECE-1 and -2 mRNA levels were significantly higher in AD than control brains. Likewise, ECE-2 protein levels were significantly elevated in AD. Conclusions: We have shown significant increases in the expression of both ECE-1 and -2 in AD brain. Our findings suggest that accumulation of A in SAD is not caused by reduced levels of ECE-1 and -2. The increased expression of these enzymes in AD may be a response to increases in A. P1-460
MATRIX METALLOPROTEINASE INHIBITION REDUCES OXIDATIVE STRESS ASSOCIATED WITH CEREBRAL AMYLOID ANGIOPATHY IN VIVO IN TRANSGENIC MICE
Monica Garcia-Alloza, Claudia Prada, Elissa M. Robbins, Rebecca A. Betensky, Steven M. Greenberg, Matthew P. Frosch, Brian J. Bacskai, Massachusetts General Hospital, Charlestown Navy Yard, MA, USA. Contact e-mail: [email protected] Background: Cerebral amyloid angiopathy (CAA), characterized by extracellular -amyloid peptide (A) deposits in the vessel walls, is present in the majority of cases of Alzheimer’s disease and is a major cause of hemorrhagic stroke. The molecular pathways activated by vascular A in causing breakdown of the vessel wall are poorly understood. Among potential candidates are A-induced synthesis, release and activation of the extracellular matrix metalloproteinases (MMP) and A-induced oxidative stress. Methods: APPswe/PS1dE9 mice (6-7 months old) and tg-2576 mice (12.5-14 months old) had permanent cranial windows placed. We used in vivo multiphoton microscopy to measure: 1) progression of CAA using methoxy-XO4, 2) activation of MMP with a green fluorescent substrate (DQ gelatin), and 3) accumulation of reactive oxygen species (ROS) with the fluorescent probe Amplex Red. Quantitative analysis of CAA burden, Amplex Red signal and MMP signal was performed using Image J and Adobe Photoshop 7. Results: Using this system, we observed that MMP activation and oxidative stress were both associated with vessel segments affected with CAA. Antioxidant treatment with N-tert-butyl-␣phenil-nitrone (PBN) was capable of reducing oxidative stress associated with CAA (⬃50% reduction, *P⬍0.001 vs. Control) without affecting MMP activation. Conversely, a selection of MMP inhibitors with different mechanisms of action, including minocycline, simvastatin and GM6001, reduced not only CAA-associated MMP activation (⬃30-40% reduction,