- Email: [email protected]
P4-330 Genistein ameliorated β-amyloid peptide (25–35)-induced hippocampal neuronal apoptosis
Poster Session P4: Therapeutics and Therapeutic Strategies - Therapeutic Strategies, Amyloid-Based
$569
of meningo-encephalitic cellular inflammatory reactions. We hypothesize that immunization with A[342 is effective but the method of vaccination should be modified to avoid the adverse reactions. Objective: To develop an effective DNA-based genetic immunization (GI) approach for treatment and prevention of AD without causing significant meningo-encephalitis. Methods: Mice were immunized for three times over 6 Weeks with a genetic vaccine plasmid encoding A[342. A gene gun was used to administer the gene vaccine to the ears. The immune response of the mice to the A[342 was monitored by enzyme-linked immunosorbent assay (ELISA), Western blot and interferon-gamma enzyme-linked immunospot (ELISPOT) assay. Results: We chemically constructed the A[~42 gene and cloned it into a GI vector. After gene gun mediated immunization in Balb/c mice a high titer of anti-human A[~42 antibodies was detected by ELISA and Western blot method. ELISPOT assays demonstrated no significant development of A[M2-specific CD8+ T cells in vaccinated mice, indicating no cellular immune response was induced. We have developed a system to break tolerance in mice and used it to produce anti-mouse A[342 antibodies in the mouse although the titer is lower than against the human AB42 peptide. We are exploring several avenues to increase the titer against self-AB42 and testing the effectiveness of this immunization in transgenic AD mice. Conclusions: Gene gun mediated genetic immunization of A[342 gene can effectively elicit a humoral immune response with no significant cell-mediated immunity against A[3 peptide. This immunotherapetuic approach could be an alternative method for therapy and prevention of AD.
Objective: To explore the feasibility of catalytic immunotherapy of AD. Methods: A panel of human Ab light chain (IgL) and polyclonal Abs isolated from the serum of human subjects was screened for cleavage of A[31-40 and A~1-42 using HPLC and mass spectroscopy. Results and conclusions: Both AI~l-40 and A[31-42 were cleaved by the IgL (clone hk14). Sequential chromatographic steps resulted in purification of the IgL to a constant level of catalytic activity, suggesting the absence of protease contaminants. Covalent complexes of the IgL with a biotinylated phosphonate diester were evident in denaturing etectrophoresis gels. As the phosphonate probe reacts selectively with activated nucleophilic residues present in serine proteases, the IgL appears to belong to this enzyme class. Mass spectroscopy suggested that A[31-40 cleavage occurred as an endopeptidase reaction at a single site, the His14-Gln15 bond, or as a sequential exopeptidase reaction resulting in cleavage of multiple sites. The cleavage pattern appears dependent on the A~ concentration, suggesting a role for differing folding and aggregation states adopted by the peptide. Polyclonal Abs isolated from the serum of human subjects also displayed Af3 peptide cleaving activity. These observations open the route towards: (a) study of Ab catalysis as a mechanism of physiological AI3 clearance; and (b) development of specific proteolytic Abs for immunotherapy of AD.
•
Iliya Lefterov* 1, Radosveta Koldamova 1, Matthias Staufenbiel 2, Milts Ikonomovic 1, Barbara Isanski 1, Steven T. DeKosky 1, John S. Lazo 1.
GENISTEIN A M E L I O R A T E D f - A M Y L O I D P E P T I D E (25-35)-INDUCED H I P P O C A M P A L N E U R O N A L APOPTOSIS
Baolu Zhao*. Institute of Biophysics, Academia Sinica, Beijing, China.
Contact e-mail: zhaobl@ sun5.ibp, ac.cn Background: In Alzheimer's disease (AD), severe neurons loss in the hippocampus and eventually leads to dementia. A few lines of evidences manifested that ~-amyloid protein (A[3) contribute a lot to the cascade by elevating intracellular free Ca 2+ and generating robust free radical and other cytotoxic stimuli. Genistein, the most active soy isoflavone, was proven to prevent diverse kinds of cell lines and primary culture from a variety of toxic stimuli. Objective(s): Protecting mechanism of genistein for neurons from apoptosis caused by A[3. Methods: We developed a model of primary hippocampal neuron apoptosis triggered by A[325-35 to approach the potential neuroprotection of genistein. Results: A[325-35induced apoptosis, confirmed by loss of mitochondrial function, neuronal DNA condensation and fragmentation, was involved with increase of intracelhilar free Ca 2+, accumulation of ROS, and activation of caspase-3. Conclusions: Our results demonstrated that, at the level of nanomolar (100 nanomolar), genistein protected neuron from injury dramatically via estrogen receptor (ER)-mediated and slightly antioxidantion; at the level of micromolar (40micromolar), genistein mainly by antioxidantion. For the first time, our study shows that genistein evidently attenuates neuronal apoptosis by various mechanisms.
•
CATALYTIC HYDROLYSIS O F A M Y L O I D ft-PEPTIDE (Aft) BY H U M A N ANTIBODIES
Hiroaki Taguchi 1, Stephanie Planque 1, Yasuhiro Nishiyama 1, Michael R. Sierks 2, Paul Szabo 3, Mark E. Weksler 3, Sudhir Paul* I. l Univ of Texas
Houston Medical School, Houston, TX, USA; 2Arizona State Univ, Tempe, AZ, USA; 3Weill Med College of Cornell Univ, New York, NY, USA. Contact e-mail: [email protected] Background: Previous studies suggest that antibodies (Abs) to amyloid beta peptide (A[3) reverse cerebral histopathology and cognitive decline in APP-transgenic mice, and the anti-A[3 Ab response correlates with arrest of cognitive decline in A[3-immunized patients with Alzheimer's disease (AD). Abs that catalyze the cleavage of A[3 can be anticipated to more effectively neutralize the neurotoxicity of A[3 compared to conventional Abs by virtue of their turnover and permanent chemical transformation capabilities.
~ L X R LIGAND TREATMENT IN VITRO AND IN VIVO IS F O L L O W E D BY ABCA1 U P R E G U L A T I O N AND D E C R E A S E IN AB S E C R E T I O N
1University of Pittsburgh, Pittsburgh, PA, USA; 2Novartis Institutes of Biomedical Research, Basel, Switzerland. Contact e-mail: [email protected] Background: Various lines of evidence indicate a link between cholesterol and AD. Recent studies in our and others laboratories have demonstrated that over expression of ABCA1 and its transcriptional upregulation, which is controlled by nuclear liver X receptors (LXR0t/[3) reduce the secretion of A[3, thus establishing a link between amyloidogenic APP processing and reverse cholesterol transport (RCT). Objective(s): Because ligand activated LXRs and their responsive genes control key metabolic processes through the amount and distribution of intracelhilar cholesterol content, they are potential therapeutic targets in AD. Methods: In continuation of our previous studies we examined amyloidogenic processing of APP in primary neurons infected with HSV-I viral vectors for transient hAPP expression and transgenic mice treated with the LXR agonist T0901317 (TO). Results: We demonstrate that amyloidogenic processing of hAPP and A~ secretion in primary neurons is substantially (>3 times) inhibited - an effect that can be reproduced by overexpressing ABCA1 in a mammalian cell model. Moreover, the application of T0 caused a significant increase in ABCA1 expression in the brain of young APP transgenic (APP23) mice and a decrease in soluble A[3. This demonstrates that it is possible to manipulate hAPP proteolytic processing and A~ secretion by the application of synthetic LXR agonists. Conclusions: Our results establish a pharmacological intervention aiming at ABCA1, or possibly other genes involved in RCT, as a promising drug development strategy for prevention and slowing the progression of AD.
•
SAFETY, TOLERABILITY, AND CHANGES IN PLASMA AND C E R E B R O S P I N A L FLUID A M Y L O I D BETA CONCENTRATIONS AFTER ADMINISTRATION OF A F U N C T I O N A L GAMMA-SECRETASE INHIBITOR IN HEALTHY VOLUNTEERS
Eric Siemers* 1, Robert A. Dean 1, Julie Satterwhite 1, Martin R. Farlow 2, Michael Skinner 1, Dan Ness 1, Patrick C. May 1. lEli Lilly and Company,
Indianapolis, IN, USA; 2Indiana University, Indianapolis, IN, USA. Contact e-mail: esiemers @lilly.corn Background: Amyloid [3 (A[3) may play a central role in the pathogenesis of Alzheimer's disease. A functional y-secretase inhibitor, LY450139, was developed that inhibits formation of A[3 in whole cell assays and transgenic
$569
of meningo-encephalitic cellular inflammatory reactions. We hypothesize that immunization with A[342 is effective but the method of vaccination should be modified to avoid the adverse reactions. Objective: To develop an effective DNA-based genetic immunization (GI) approach for treatment and prevention of AD without causing significant meningo-encephalitis. Methods: Mice were immunized for three times over 6 Weeks with a genetic vaccine plasmid encoding A[342. A gene gun was used to administer the gene vaccine to the ears. The immune response of the mice to the A[342 was monitored by enzyme-linked immunosorbent assay (ELISA), Western blot and interferon-gamma enzyme-linked immunospot (ELISPOT) assay. Results: We chemically constructed the A[~42 gene and cloned it into a GI vector. After gene gun mediated immunization in Balb/c mice a high titer of anti-human A[~42 antibodies was detected by ELISA and Western blot method. ELISPOT assays demonstrated no significant development of A[M2-specific CD8+ T cells in vaccinated mice, indicating no cellular immune response was induced. We have developed a system to break tolerance in mice and used it to produce anti-mouse A[342 antibodies in the mouse although the titer is lower than against the human AB42 peptide. We are exploring several avenues to increase the titer against self-AB42 and testing the effectiveness of this immunization in transgenic AD mice. Conclusions: Gene gun mediated genetic immunization of A[342 gene can effectively elicit a humoral immune response with no significant cell-mediated immunity against A[3 peptide. This immunotherapetuic approach could be an alternative method for therapy and prevention of AD.
Objective: To explore the feasibility of catalytic immunotherapy of AD. Methods: A panel of human Ab light chain (IgL) and polyclonal Abs isolated from the serum of human subjects was screened for cleavage of A[31-40 and A~1-42 using HPLC and mass spectroscopy. Results and conclusions: Both AI~l-40 and A[31-42 were cleaved by the IgL (clone hk14). Sequential chromatographic steps resulted in purification of the IgL to a constant level of catalytic activity, suggesting the absence of protease contaminants. Covalent complexes of the IgL with a biotinylated phosphonate diester were evident in denaturing etectrophoresis gels. As the phosphonate probe reacts selectively with activated nucleophilic residues present in serine proteases, the IgL appears to belong to this enzyme class. Mass spectroscopy suggested that A[31-40 cleavage occurred as an endopeptidase reaction at a single site, the His14-Gln15 bond, or as a sequential exopeptidase reaction resulting in cleavage of multiple sites. The cleavage pattern appears dependent on the A~ concentration, suggesting a role for differing folding and aggregation states adopted by the peptide. Polyclonal Abs isolated from the serum of human subjects also displayed Af3 peptide cleaving activity. These observations open the route towards: (a) study of Ab catalysis as a mechanism of physiological AI3 clearance; and (b) development of specific proteolytic Abs for immunotherapy of AD.
•
Iliya Lefterov* 1, Radosveta Koldamova 1, Matthias Staufenbiel 2, Milts Ikonomovic 1, Barbara Isanski 1, Steven T. DeKosky 1, John S. Lazo 1.
GENISTEIN A M E L I O R A T E D f - A M Y L O I D P E P T I D E (25-35)-INDUCED H I P P O C A M P A L N E U R O N A L APOPTOSIS
Baolu Zhao*. Institute of Biophysics, Academia Sinica, Beijing, China.
Contact e-mail: zhaobl@ sun5.ibp, ac.cn Background: In Alzheimer's disease (AD), severe neurons loss in the hippocampus and eventually leads to dementia. A few lines of evidences manifested that ~-amyloid protein (A[3) contribute a lot to the cascade by elevating intracellular free Ca 2+ and generating robust free radical and other cytotoxic stimuli. Genistein, the most active soy isoflavone, was proven to prevent diverse kinds of cell lines and primary culture from a variety of toxic stimuli. Objective(s): Protecting mechanism of genistein for neurons from apoptosis caused by A[3. Methods: We developed a model of primary hippocampal neuron apoptosis triggered by A[325-35 to approach the potential neuroprotection of genistein. Results: A[325-35induced apoptosis, confirmed by loss of mitochondrial function, neuronal DNA condensation and fragmentation, was involved with increase of intracelhilar free Ca 2+, accumulation of ROS, and activation of caspase-3. Conclusions: Our results demonstrated that, at the level of nanomolar (100 nanomolar), genistein protected neuron from injury dramatically via estrogen receptor (ER)-mediated and slightly antioxidantion; at the level of micromolar (40micromolar), genistein mainly by antioxidantion. For the first time, our study shows that genistein evidently attenuates neuronal apoptosis by various mechanisms.
•
CATALYTIC HYDROLYSIS O F A M Y L O I D ft-PEPTIDE (Aft) BY H U M A N ANTIBODIES
Hiroaki Taguchi 1, Stephanie Planque 1, Yasuhiro Nishiyama 1, Michael R. Sierks 2, Paul Szabo 3, Mark E. Weksler 3, Sudhir Paul* I. l Univ of Texas
Houston Medical School, Houston, TX, USA; 2Arizona State Univ, Tempe, AZ, USA; 3Weill Med College of Cornell Univ, New York, NY, USA. Contact e-mail: [email protected] Background: Previous studies suggest that antibodies (Abs) to amyloid beta peptide (A[3) reverse cerebral histopathology and cognitive decline in APP-transgenic mice, and the anti-A[3 Ab response correlates with arrest of cognitive decline in A[3-immunized patients with Alzheimer's disease (AD). Abs that catalyze the cleavage of A[3 can be anticipated to more effectively neutralize the neurotoxicity of A[3 compared to conventional Abs by virtue of their turnover and permanent chemical transformation capabilities.
~ L X R LIGAND TREATMENT IN VITRO AND IN VIVO IS F O L L O W E D BY ABCA1 U P R E G U L A T I O N AND D E C R E A S E IN AB S E C R E T I O N
1University of Pittsburgh, Pittsburgh, PA, USA; 2Novartis Institutes of Biomedical Research, Basel, Switzerland. Contact e-mail: [email protected] Background: Various lines of evidence indicate a link between cholesterol and AD. Recent studies in our and others laboratories have demonstrated that over expression of ABCA1 and its transcriptional upregulation, which is controlled by nuclear liver X receptors (LXR0t/[3) reduce the secretion of A[3, thus establishing a link between amyloidogenic APP processing and reverse cholesterol transport (RCT). Objective(s): Because ligand activated LXRs and their responsive genes control key metabolic processes through the amount and distribution of intracelhilar cholesterol content, they are potential therapeutic targets in AD. Methods: In continuation of our previous studies we examined amyloidogenic processing of APP in primary neurons infected with HSV-I viral vectors for transient hAPP expression and transgenic mice treated with the LXR agonist T0901317 (TO). Results: We demonstrate that amyloidogenic processing of hAPP and A~ secretion in primary neurons is substantially (>3 times) inhibited - an effect that can be reproduced by overexpressing ABCA1 in a mammalian cell model. Moreover, the application of T0 caused a significant increase in ABCA1 expression in the brain of young APP transgenic (APP23) mice and a decrease in soluble A[3. This demonstrates that it is possible to manipulate hAPP proteolytic processing and A~ secretion by the application of synthetic LXR agonists. Conclusions: Our results establish a pharmacological intervention aiming at ABCA1, or possibly other genes involved in RCT, as a promising drug development strategy for prevention and slowing the progression of AD.
•
SAFETY, TOLERABILITY, AND CHANGES IN PLASMA AND C E R E B R O S P I N A L FLUID A M Y L O I D BETA CONCENTRATIONS AFTER ADMINISTRATION OF A F U N C T I O N A L GAMMA-SECRETASE INHIBITOR IN HEALTHY VOLUNTEERS
Eric Siemers* 1, Robert A. Dean 1, Julie Satterwhite 1, Martin R. Farlow 2, Michael Skinner 1, Dan Ness 1, Patrick C. May 1. lEli Lilly and Company,
Indianapolis, IN, USA; 2Indiana University, Indianapolis, IN, USA. Contact e-mail: esiemers @lilly.corn Background: Amyloid [3 (A[3) may play a central role in the pathogenesis of Alzheimer's disease. A functional y-secretase inhibitor, LY450139, was developed that inhibits formation of A[3 in whole cell assays and transgenic