- Email: [email protected]
S2-03-06 NSAID modulators of the gamma secretase: high throughput tools and mechanistic insights
Symposium $2-04: Therapeutic Strategies fragments). Upon successful assembly the complex is released from early compartments to be targeted to the plasma membrane. PS itself contains an ER retention signal, which prevents forward transport in the absence of gamma-secretase complex formation. In addition, we identified a binding site within PS1, which mediates the interaction with other gamma-secretase complex components.
$2-03-04 ] ASSEMBLY OF THE GAMMA-SECRETASE COMPLEX Peter St George-Hyslop* 1, Fusheng Chen 1, YongJun Gu 1, Hiroshi Hasegawa 1 Agnes Petit 1, Anurag Tandon 1, Peter Mastrangelo 1, Taiichi Katayama 1, Paul Matthews 2, Stephen Schmidt 2, David Westaway 1, Gerold Schmitt-Ulins I , Paul E. Fraser 1. I University of Toronto, Toronto,
ON, Canada; 2Nathan Kline Institute, New York University School of Medicine, Orangeburg, NY,, USA. Contact e-mail: [email protected]
Background: The presenilin proteins are homologous polytopic, transmembrahe proteins that are necessary for the endoproteolytic cleavage of several Type 1 transmembrane proteins including the amyloid precursor protein (APP). This latter cleavage generates Abeta and a series of C-terminal stubs (epsilon-stubs). Several other proteins including nicastrin, APH-1, and PEN2 are required for this activity. However, the precise mechanisms of both the presenilin-dependant cleavage and of the assembly and maturation of the presenilins into high molecular weight heteromeric protein complexes remain obscure. Objectives: To understand the molecular biology of the assembly and activation ofpresenilin complexes. Methods: Combinations of immunocytochemical, biochemical and cell biological methods including glycerol velocity gradient and 2-D Blue Native gel electrophoresis were used to investigate the assembly, maturation, and activation of Presenilin complexes. Site directed mutagenesis was employed to identify functionally important residues. Results: These studies reveal that there are several distinct presenilin complexes including a series of non-functional complexes between 134kDa and 440kDa. However, the complexes with highest specific activity have molecular weights of greater than 770kDa. Several conserved residues within APH-1 and PEN-2 appear to be important in the normal assembly and maturation of complexes. Conclusions: These studies establish that the presenilin complexes are assembled from a series of smaller complexes. The stoichiometry and componentry of the higher molecular weight functionally active complexes which are functionally active will be described.
S2-03-05 ] NOVEL C H O L E S T E R O L - B A S E D R E G U L A T I O N OF 13- AND y-SECRETASE ACTIVITIES Dora M. Kovacs*. Massachusetts General Hospital, Boston (Charlestown),
MA, USA. Contact e-mail: [email protected] Increasing evidence suggests a role for cholesterol on secretase activities. Therapies already developed for dyslipidemia and atherosclerosis are gaining attention as attractive strategies for also reducing A[3 generation. Cellular and animal studies have shown that HMG-CoA reductase inhibitors, or statins, are effective in lowering both cholesterol and A[3 generation, while clinical studies suggest that statins partially stabilize cognitive function in AD patients. Non-stalin therapies are now being developed for use as monotherapy or in combination with statins. These include acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors, which convert cholesterol and fatty acids into cholesteryl-esters. A well-characterized ACAT inhibitor, CP-113,818, inhibits At3 production in cultured cells by affecting processing of APP by both ~3- and y-secretases, while also inhibiting cz-secretase activity (Puglielli et al., 2001). We have now assessed the efficacy of CP-113,818 in reducing AD-like pathology in the brains of transgenic mice expressing human APP751 containing the London (V717I) and Swedish (K670M/N671L) mutations. Two months of treatment with CP113,818 decreased brain and liver cholesteryl-esters by up to 86 and 93%, respectively. Remarkably, amyloid plaque load was reduced by 88-99%, plaque coverage by up to 97%, and insoluble AI3 levels by 83-96%. Additionally, CP-113,818 improved spatial learning, which correlated with decreased AlL The levels of C99 (APP C-terminal fragment produced by
$29
[3-secmtase) and C83 (APP C-terminal fragment produced by c~-secretase) were unchanged, while secreted APP fragments (produced by both c~- and 13-secretases) were decreased by ~60%, suggesting that all three secretase activities are inhibited in CP-113,818-treated brains. In cholesterol-mutant cells lacking ACAT activity, APP is steered toward a novel proteolytic pathway that replaces both el- and the amyloidogenic [~-cleavages of APP. This alternative pathway involves a cleavage at Glu281 and tightly correlates with reduced ACAT activity and AI3 generation. Our results suggest that ACAT inhibitors, alone or in combination with statins, may be considered as a novel strategy for the treatment and prevention of Alzheimer's disease based on reduced secretase activity.
$2-03-06 l NSAID MODULATORS OF THE GAMMA SECRETASE: HIGH THROUGHPUT TOOLS AND MECHANISTIC INSIGHTS Jeffrey S. Nye*. Johnson and Johnson, PRD, Titusville, NJ, USA. Contact
e-mail: JNye@ PRDUS.JNJ.com
Background: Recent studies have shown that a subset of NSAIDs preferentially reduce the production of A[31-42 and induce A131-38, while falling to inhibit Notch cleavage (Weggen, Nature 2001). Objective: Potent compounds that modulate the activity of the gamma secretase in this manner may have advantages over classical gamma secretase inhibitors for Atzheimer's disease therapeutics. Methods: To facilitate drug discovery efforts, we developed a high-throughput AIM-38 assay and reportcr-transfection assays to measure the epsilon-site cleavage of Notch and APP. Results: Use of these and other assays has led to a pharmacologic classification of NSAIDs and 7-secretase inhibitors into active modulators, reverse modulators, inhibitors, and inactive compounds. At the y-site, modulators show induction of A[3138 in the same concentration range as their inhibition of At31-42 production. Cleavage at the s-site of Notch, APP and Erb-B4 was preferentially spared by modulators compared to their efficacy at blocking AI31-42 production while inhibitors blocked both ~-site and "/-site cleavages. Conclusions: These observations suggest that the mechanism of action for modulators of the y-secretase involves selective action at y-site cleavages with relative sparing of e-site cleavages for any substrate. These studies are also consistent with a model in which modulators of the gamma secretase shift cleavage of the 42-43 bond to cleavage of the 38-39 bond by an unknown mechanism.
S y m p o s i u m $2-04: Therapeutic Strategies THE THERAPEUTIC POTENTIAL OF M1 I$2-04-01 ] MUSCARINIC AGON1STS IN A L Z H E I M E R ' S DISEASE (AD) AND CEREBRAL A M Y L O I D ANGIOPATHY (CAA) Abraham Fisher* 1, Zipora Pittel I, Rachel Brandeis l, Thomas G. Beach 2, D. Larry Sparks 2, Tobias Hartmann 3, Ginny G. Farias 4, Noelle Bons 5, Nira Bar-Ner 1, Nibaldo C. Inestrosa 4. 1Israel Institute for Biological
Research, Ness Ziona, Israel; 2Sun Health Research Center, Sun City, AZ, USA; 3University of Heidelberg, Heidlberg, Germany; 4 Universita Catholica, Santiago, Chile; 5E.PH.E, Montpellier, France. Contact e-mail: fisher_a @netvision, net.il The M1 muscarinic receptor (M1 mAChR), known to mediate cognition, is relatively spared in AD and is a rational target for treatment of cognitive impairments. Some muscarinic agonists were effective on cognition and psychosis in AD patients. However, their clinical value is limited by lack of selectivity on M1 mAChR, by adverse effects and overdosing, as the clinical studies were governed by the pharmacoldnetic rather than the pharmacodynamic profile of such agonists on memory. The M1 agonists AF102B [Cevimeline], AF150(S) and AF267B can beneficially modulate three major hallmarks of AD: cognitive functions in several animal models of AD and with a wide safety margin, A[3 and tan hyperphosphorylation (TAU-P). Thus M1 mAChR-activation by M1 agonists - increased ~APPs (via activation of PKC, MAPK, ct-secretase), decreased AI3 (probably via 6-
$2-03-04 ] ASSEMBLY OF THE GAMMA-SECRETASE COMPLEX Peter St George-Hyslop* 1, Fusheng Chen 1, YongJun Gu 1, Hiroshi Hasegawa 1 Agnes Petit 1, Anurag Tandon 1, Peter Mastrangelo 1, Taiichi Katayama 1, Paul Matthews 2, Stephen Schmidt 2, David Westaway 1, Gerold Schmitt-Ulins I , Paul E. Fraser 1. I University of Toronto, Toronto,
ON, Canada; 2Nathan Kline Institute, New York University School of Medicine, Orangeburg, NY,, USA. Contact e-mail: [email protected]
Background: The presenilin proteins are homologous polytopic, transmembrahe proteins that are necessary for the endoproteolytic cleavage of several Type 1 transmembrane proteins including the amyloid precursor protein (APP). This latter cleavage generates Abeta and a series of C-terminal stubs (epsilon-stubs). Several other proteins including nicastrin, APH-1, and PEN2 are required for this activity. However, the precise mechanisms of both the presenilin-dependant cleavage and of the assembly and maturation of the presenilins into high molecular weight heteromeric protein complexes remain obscure. Objectives: To understand the molecular biology of the assembly and activation ofpresenilin complexes. Methods: Combinations of immunocytochemical, biochemical and cell biological methods including glycerol velocity gradient and 2-D Blue Native gel electrophoresis were used to investigate the assembly, maturation, and activation of Presenilin complexes. Site directed mutagenesis was employed to identify functionally important residues. Results: These studies reveal that there are several distinct presenilin complexes including a series of non-functional complexes between 134kDa and 440kDa. However, the complexes with highest specific activity have molecular weights of greater than 770kDa. Several conserved residues within APH-1 and PEN-2 appear to be important in the normal assembly and maturation of complexes. Conclusions: These studies establish that the presenilin complexes are assembled from a series of smaller complexes. The stoichiometry and componentry of the higher molecular weight functionally active complexes which are functionally active will be described.
S2-03-05 ] NOVEL C H O L E S T E R O L - B A S E D R E G U L A T I O N OF 13- AND y-SECRETASE ACTIVITIES Dora M. Kovacs*. Massachusetts General Hospital, Boston (Charlestown),
MA, USA. Contact e-mail: [email protected] Increasing evidence suggests a role for cholesterol on secretase activities. Therapies already developed for dyslipidemia and atherosclerosis are gaining attention as attractive strategies for also reducing A[3 generation. Cellular and animal studies have shown that HMG-CoA reductase inhibitors, or statins, are effective in lowering both cholesterol and A[3 generation, while clinical studies suggest that statins partially stabilize cognitive function in AD patients. Non-stalin therapies are now being developed for use as monotherapy or in combination with statins. These include acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors, which convert cholesterol and fatty acids into cholesteryl-esters. A well-characterized ACAT inhibitor, CP-113,818, inhibits At3 production in cultured cells by affecting processing of APP by both ~3- and y-secretases, while also inhibiting cz-secretase activity (Puglielli et al., 2001). We have now assessed the efficacy of CP-113,818 in reducing AD-like pathology in the brains of transgenic mice expressing human APP751 containing the London (V717I) and Swedish (K670M/N671L) mutations. Two months of treatment with CP113,818 decreased brain and liver cholesteryl-esters by up to 86 and 93%, respectively. Remarkably, amyloid plaque load was reduced by 88-99%, plaque coverage by up to 97%, and insoluble AI3 levels by 83-96%. Additionally, CP-113,818 improved spatial learning, which correlated with decreased AlL The levels of C99 (APP C-terminal fragment produced by
$29
[3-secmtase) and C83 (APP C-terminal fragment produced by c~-secretase) were unchanged, while secreted APP fragments (produced by both c~- and 13-secretases) were decreased by ~60%, suggesting that all three secretase activities are inhibited in CP-113,818-treated brains. In cholesterol-mutant cells lacking ACAT activity, APP is steered toward a novel proteolytic pathway that replaces both el- and the amyloidogenic [~-cleavages of APP. This alternative pathway involves a cleavage at Glu281 and tightly correlates with reduced ACAT activity and AI3 generation. Our results suggest that ACAT inhibitors, alone or in combination with statins, may be considered as a novel strategy for the treatment and prevention of Alzheimer's disease based on reduced secretase activity.
$2-03-06 l NSAID MODULATORS OF THE GAMMA SECRETASE: HIGH THROUGHPUT TOOLS AND MECHANISTIC INSIGHTS Jeffrey S. Nye*. Johnson and Johnson, PRD, Titusville, NJ, USA. Contact
e-mail: JNye@ PRDUS.JNJ.com
Background: Recent studies have shown that a subset of NSAIDs preferentially reduce the production of A[31-42 and induce A131-38, while falling to inhibit Notch cleavage (Weggen, Nature 2001). Objective: Potent compounds that modulate the activity of the gamma secretase in this manner may have advantages over classical gamma secretase inhibitors for Atzheimer's disease therapeutics. Methods: To facilitate drug discovery efforts, we developed a high-throughput AIM-38 assay and reportcr-transfection assays to measure the epsilon-site cleavage of Notch and APP. Results: Use of these and other assays has led to a pharmacologic classification of NSAIDs and 7-secretase inhibitors into active modulators, reverse modulators, inhibitors, and inactive compounds. At the y-site, modulators show induction of A[3138 in the same concentration range as their inhibition of At31-42 production. Cleavage at the s-site of Notch, APP and Erb-B4 was preferentially spared by modulators compared to their efficacy at blocking AI31-42 production while inhibitors blocked both ~-site and "/-site cleavages. Conclusions: These observations suggest that the mechanism of action for modulators of the y-secretase involves selective action at y-site cleavages with relative sparing of e-site cleavages for any substrate. These studies are also consistent with a model in which modulators of the gamma secretase shift cleavage of the 42-43 bond to cleavage of the 38-39 bond by an unknown mechanism.
S y m p o s i u m $2-04: Therapeutic Strategies THE THERAPEUTIC POTENTIAL OF M1 I$2-04-01 ] MUSCARINIC AGON1STS IN A L Z H E I M E R ' S DISEASE (AD) AND CEREBRAL A M Y L O I D ANGIOPATHY (CAA) Abraham Fisher* 1, Zipora Pittel I, Rachel Brandeis l, Thomas G. Beach 2, D. Larry Sparks 2, Tobias Hartmann 3, Ginny G. Farias 4, Noelle Bons 5, Nira Bar-Ner 1, Nibaldo C. Inestrosa 4. 1Israel Institute for Biological
Research, Ness Ziona, Israel; 2Sun Health Research Center, Sun City, AZ, USA; 3University of Heidelberg, Heidlberg, Germany; 4 Universita Catholica, Santiago, Chile; 5E.PH.E, Montpellier, France. Contact e-mail: fisher_a @netvision, net.il The M1 muscarinic receptor (M1 mAChR), known to mediate cognition, is relatively spared in AD and is a rational target for treatment of cognitive impairments. Some muscarinic agonists were effective on cognition and psychosis in AD patients. However, their clinical value is limited by lack of selectivity on M1 mAChR, by adverse effects and overdosing, as the clinical studies were governed by the pharmacoldnetic rather than the pharmacodynamic profile of such agonists on memory. The M1 agonists AF102B [Cevimeline], AF150(S) and AF267B can beneficially modulate three major hallmarks of AD: cognitive functions in several animal models of AD and with a wide safety margin, A[3 and tan hyperphosphorylation (TAU-P). Thus M1 mAChR-activation by M1 agonists - increased ~APPs (via activation of PKC, MAPK, ct-secretase), decreased AI3 (probably via 6-