- Email: [email protected]
Reverse chemical genetics approach to identify druggable gene products in Alzheimer's disease
Symposium S2-03: Disease Mechanisms-APP/A Beta in relation to MCI conversion differed among clinical variables: Deceleration in gait speed occurred over a decade before the transition to MCI, followed by the acceleration in decline in neuropsychological tests and expansion in ventricular volume, the latter occurring about 3 to 4 years before the transition. Our exploratory analysis using quantile regression models, which focuses on multi-factorial changes within individuals, appears to be a useful and valid method to elucidate potential physiological mechanisms occurring during the pre-symptomatic phase of dementia. Conclusions: Understanding the order and combinations of intra-individual changes associated with transition to MCI is fundamental to recognition of meaningful change and the pathophysiological pathways underlying developing dementia. These data can ultimately be applied over time to help clinicians in the early identification of dementia and to institute early treatment strategies. MONDAY, JULY 12, 2010 SYMPOSIUM S2-03 DISEASE MECHANISMS-APP/A BETA S2-03-01
SPLICING AND DICING IN APP PROCESSING: TARGETING PROTEASES AND MRNAS THAT REGULATE A-BETA PRODUCTION
Michael S. Wolfe, Harvard Medical School, Boston, MA, USA. Contact e-mail: [email protected] Background: Beta- and gamma-secretases catalyze production of A-beta, the major protein component of the cerebral plaques of Alzheimer’s disease, from its precursor protein (APP). While these enzymes are both aspartyl proteases and top therapeutic targets, their biochemical properties are quite different, and each presents different problems for drug discovery. Gammasecretase inhibitors should avoid blocking proteolysis of the Notch receptor, part of an essential signaling pathway in cell differentiation. Beta-secretase (BACE1) inhibitors have poor drug-like properties and do not access the brain well. OBJECTIVES: To develop Notch-sparing gamma-secretase inhibitors and to explore the regulation of mRNA splicing of BACE1 as an alternative strategy toward lowering its activity. Methods: From initial hits, iterative design, synthesis and biochemical evaluation was carried out to identify potent and selective Notch-sparing gamma-secretase inhibitors. Promising compounds were further tested in cell culture for A-beta-lowering effects and evaluated for physiochemical and pharmacokinetic properties. BACE1 alternative splicing was studied using quantitative and semi-quantitative RT-PCR methods and via a mini-gene system that recapitulates endogenous BACE1 splicing. The A-beta-generating activity of BACE1 isoforms was determined as well. Results: Iterative medicinal chemistry has led to gamma-secretase inhibitors that effectively block A-beta production and that are substantially selective with respect to Notch proteolysis. These compounds have promising physicochemical and pharmacokinetic properties, suggesting that further preclinical development is warranted. Alternative splicing of BACE1 results in isoforms with little proteolytic activity, and shunting splicing of BACE1 toward these isoforms with antisense oligonucleotides results in reduced A-beta production in cells. Cis-elements in the BACE1 pre-mRNA that regulate alternative splicing have been identified, raising the prospect of finding small molecule modulators of BACE1 splicing. Conclusions: The discovery of Notch-sparing inhibitors makes gammasecretase a much more promising therapeutic target. Beta-secretase activity can be reduced indirectly, at the level of pre-mRNA splicing, by shunting activity to inactive isoforms. S2-03-02
MOLECULAR MECHANISM OF ACTION OF GAMMA-SECRETASE INHIBITORS AND MODULATORS
Taisuke Tomita, The University of Tokyo, Tokyo, Japan. Contact e-mail: [email protected] Background: Genetic and biological studies provide strong evidence that the production and deposition of amyloid-beta peptides (Abeta) contribute to the etiology of Alzheimer’s disease (AD). Thus, gamma-secretase, that is a responsible protease for the Abeta generation, is a plausible molecular
S93
target for AD treatment. gamma-Secretase is an unusual aspartic protease that cleaves the scissile bond within the transmembrane domain. This unusual enzyme is composed of a high molecular weight membrane protein complex containing presenilin, nicastrin, Aph-1 and Pen-2. Presenilin, a catalytic subunit for the gamma-secretase, forms a ‘‘catalytic pore’’ structure in which intramembrane cleavage might be occurred. Drugs that regulate the production of Abeta by inhibiting or modulating the gamma-secretase activity could provide a disease-modifying effect on AD, although recent studies suggest that the gamma-secretase plays important roles in cellular signaling including Notch pathway. Thus, much attention is now focused on the understanding the molecular mechanism whereby the gamma-secretase inhibitors and modulators specifically regulate the Abeta-generating gamma-secretase activity. Methods: Using chemical biological technique (i.e., photoaffinity labeling), we have identified direct molecular targets of the gamma-secretase inhibitors and modulators. We have also analyzed the effects of these compounds on the catalytic pore structure using biochemical structural analysis. Results: We found that the Notch-sparing gamma-secretase inhibitor and modulator directly target the presenilin and affect the structure of the catalytic pore. Conclusions: Our results clearly indicate that presenilin is an important molecular target for the Notch-sparing gamma-secretase inhibitors as well as modulators. I will also summarize our recent studies on the gammasecretase complex, and envision the direction for developing the effective and selective gamma-secretase inhibitors as therapeutics for AD. S2-03-03
ROLE OF PRESENILIN 1 IN ADULT NEUROGENESIS
Sangram Sisodia, University of Chicago, Chicago, IL, USA. Contact e-mail: [email protected] Background: Inheritance of mutant genes encoding presenilins (PS1 and PS2) variants cause autosomal dominant, familial Alzheimer’s disease (FAD). We have investigated the influence of expressing these mutant PS1 variants on hippocampal neurogenesis in transgenic mouse models and have shown that expression of mutant PS1 leads to impairments in environmental enrichment (EE)-mediated proliferation and differentiation of hippocampal progenitor cells (NPCs). Methods: Mouse prion promoter-driven transgenes encoding either human wild-type PS1 or FAD-linked M146L and aE9 PS1 variants were exposed to standard housing conditions, or to EE, a setting that is known to induce proliferation of progenitors (NPCs) in the dentate subgranular zone and differentiation of these cells towards a neurogenic fate. In contrast to standard housing conditions, in which NPC proliferation, survival and neuronal differentiation is unaffected by expression of either wtPS1 or PS1 variants, these parameters are markedly attenuated in the hippocampus of mice expressing mutant PS1 following EE. We have shown that the proliferation and neurogenic potential of NPCs expressing either wt or mutant PS1 are indistinguishable in vitro, but that the conditioned media from microglia expressing mutant PS1 impairs the proliferation and neuronal lineage commitment of NPCs expressing wtPS1. Results: Using proteomic and high density microrray strategies, we show that the levels of secreted chemokines in the conditioned medium of microglia expressing mutant PS1 mutant, or in microglia from enriched adult mice are significantly altered and we argue that these factors are, at least in part, responsible for the defects in proliferation and neurogenesis observed in vivo. Conclusions: Current efforts are focused on the purification and molecular analysis of microglia from enriched mice and the development of a conditional transgenic mouse model that will allow us to evaluate the impact of specific cell types in the CNS on mutant PS1-mediated alterations in NPC proliferation and neurogenesis in the hippocampus. These findings support a non-cell-autonomous role for mutant PS1 in hippocampal neurogenesis. S2-03-04
REVERSE CHEMICAL GENETICS APPROACH TO IDENTIFY DRUGGABLE GENE PRODUCTS IN ALZHEIMER’S DISEASE
Tae-Wan Kim, Columbia University, New York, NY, USA. Contact e-mail: [email protected] Background: The amyloid beta-peptide (Abeta) is produced by sequential proteolytic cleavage of beta-amyloid precursor protein (APP) by a set of
S94
Focus Topics FT-02: ADNI, What’s Next? State-of-the-Art Methods for Exploring CNS Developmental and Neurodegenerative Disorders
membrane-bound proteases termed beta-secretase (BACE1) and g-secretase. The level of Abeta in the brain is critically associated with both pathological and behavioral/clinical phenotypes of AD. Thus, CNS penetrable small molecules that can reduce Abeta in an effective and safe manner remains one of the most promising approaches for the treatment of AD. While a number of direct small molecule inhibitors of beta- or g-secretase have been developed, many of them have suffered side effects owing to their target-associated toxicity. Methods: In the present study, we conducted reverse chemical genetic screening to identify small molecule inhibitors of BACE1-mediated cleavage of APP with a goal to ultimately identify a putative protein target of the identified small molecule inhibitor. In order to identify inhibitor compounds that act via a novel mechanism, we developed a cell-based assay in intact neuronal cells, based on antibody-mediated specific capture of the BACE1-derived secreted APP ectodomain (sAPPbeta) fused to a secreted alkaline phosphatase (SEAP) reporter. Results: By screening small molecule libraries using this cell system, we identified small molecules that can inhibit BACE1-mediated cleavage of APP without directly interfering with BACE1 activity. One such compound was subjected to medicinal chemistry to improve the bioactivity and the resulting compound (termed C2) reduces Abeta levels in cultured primary neurons as well as a mouse model of AD. We are currently investigating a putative cellular target for this compound. Conclusions: Identification of a small molecule modifier of the Abeta generation pathway and uncovering its cellular target may serve as the foundation for development of new therapeutic agents that could selectively probe pathological, but not normal, function of the Abeta-producing enzymes, such as BACE1. S2-03-05
IN VIVO TARGETING OF ABETA AND TAU AMYLOIDOGENESES, NEUROINFLAMMATION, CALCIUM-MEDIATED NEUROTOXICITY AND THEIR INTERPLAYS
Makoto Higuchi, National Institute of Radiological Sciences, Chiba, Japan. Contact e-mail: [email protected] Background: Pathological accumulations of amyloid beta peptide (Abeta) and tau aggregates are critically implicated in Alzheimer’s disease (AD) as initiators of the neurodegenerative cascade, while it is yet to be elucidated how these amyloidoses and downstream key processes interplay to be embodied in a self-perpetuating cycle leading to neurotoxicities. We here employed in vivo molecular imaging techniques for genetically engineered mice modeling AD to identify neurotoxic subspecies of Abeta and tau assemblies and their reciprocal links to neuroinflammation and other intermediate events in the cascade. Methods: Depositions of Abeta and tau fribrils in transgenic (Tg) mice expressing mutant amyloid precursor protein (APP) and tau were assayed by positron emission tomography (PET) with specific radioligands. Neurotoxic inflammation was visualized in these animals using PET tracers for 18-kDa translocator protein (TSPO). Results: Formation of extracellular amyloid lesions enriched with N-terminally truncated and cyclyzed Abeta, AbetaN3pE, in APP Tg mice induced amyloid PET signals characteristic of AD brains, and was enhanced by knock-down of an Abeta-degrading endopeptidase, neprilysin. Meanwhile, Abeta aggregates in hippocampal neurons undetectable by amyloid PET strongly provoked TSPO-positive microgliosis and neuronal loss. Similarly, tau fibrillogenesis prior to the emergence of PET-positive inclusions in the hippocampus of tau Tg mice gave rise to massive TSPO signals and neuronal death. This TSPO-positive neuroinflammation was accompanied by upregulation of an AbetaN3pE-producing enzyme, glutaminyl cyclase (QC), indicating interlinks among Abeta and tau depositions and toxic inflammation mediated by QC. Circumvention of excessive TSPO rises led to effective removals of Abeta and tau assemblies in immunological treatments of Tg mice with cloned microglial implants and immunizations for these molecules. Among diverse amyloid-associated events investigated here, activation of calcium-dependent proteases, calpains, was demonstrated to influence the Abeta deposition and concurrent TSPO-positive gliosis by genetically manipulating levels of a natural calpain inhibitor, calpastatin, in APP Tg mice. Conclusions: Our data illustrate crucial roles of specific proteolytic pathways in the mutual crosstalk among amyloidogen-
esis, neurotoxic glia and disrupted calcium homeostasis in animal models. Low-order multimerization of Abeta and tau may trigger neurodegenerative deteriorations, and this notion would be tested in humans with the aid of radiotracers utilized in the present study. S2-03-06
MOLECULAR DETERMINANTS OF ABETA TOXICITY
Roberto Cappai, The University of Melbourne, Melbourne, Australia. Contact e-mail: [email protected] Background: The Alzheimer’s disease Amyloid Precursor Protein is sequentially processed by beta and gamma-secretase to yield the neurotoxic Amyloid-beta (Abeta) peptide, the principal component of the senile plaques in Alzheimer’s disease brains. Abeta is a 40- 42 amino acid peptide that binds copper (Cu) and this interaction with Cu can modulate Abeta’s neurotoxic activity. The molecular basis for the Abeta and copper interaction and how it affects Abeta toxicity is being delineated. Methods: Primary mouse neuronal cortical cultures are used as the main cell based model to measure Abeta toxicity. An additional model of toxicity we use is inhibition of long term potentiation of mouse hippocampal slices. Results: The Cu:Abeta ratio modulates the Abeta folding-aggregation pathway and its neurotoxic activity. Abeta toxicity does not correlate with the ability to form amyloid, but it does did correlate with lipid peroxidation and dityrosine formation. Our sequence-activity studies, with a range of mutant peptides, have identified the histidine and tyrosine residues as being important for modulating Abeta:Cu chemistry, Abeta oligomerisation and membrane binding. We have identified phosphatidylserine as a cellular target that binds Abeta, and mediates Abeta toxicity and Abeta inhibition of long term potentiation. We are currently investigating the cell signaling pathways affected by Abeta and the role of ancillary proteins on Abeta cell binding and Abeta toxicity. Conclusions: Our current understanding of the molecular determinants of Abeta:Cu mediated toxicity is leading to the development of novel therapeutic approaches that target Abeta and inhibit either Abeta cell binding and/or Abeta toxicity. MONDAY, JULY 12, 2010 FOCUS TOPICS FT-02 ADNI, WHAT’S NEXT? STATE-OF-THE-ART METHODS FOR EXPLORING CNS DEVELOPMENTAL AND NEURODEGENERATIVE DISORDERS FT-02-01
ADNI: KEY ELEMENTS TO SUCCESS
Neil Buckholtz, National Institute on Aging, Bethesda, MD, USA. Contact e-mail: [email protected] This presentation with feature a review of the key elements that led to the successful partnerships and scientific breakthroughs made possible by ADNI. It will examine how these elements may assist in the establishment of future multi-sector efforts. FT-02-02
LEVERAGING THE PRIVATE SECTOR
Steve Paul, Lilly Research Labs, Indianapolis, IN, USA. Contact e-mail: [email protected] The significant investment from both the public and private sector was critical for the success of ADNI. This presentation will examine how similar public-private partnerships can be leveraged to expand the efforts of ADNI to focus on a broader array of neurological disorders. FT-02-03
LEVERAGING FUTURE TECHNOLOGIES
Belinda Seto, National Institute of Biomedical Imaging and Bioengineering, Bethesda, MD, USA. Contact e-mail: [email protected] Advances in imaging technologies have been critical to the success of ADNI. As efforts are undertaken to move forward and expand to other disorders of the nervous system additional technologies will be necessary. This talk will examine the future trends in technology development including neuroimaging, and how this can help shape the future efforts.
SPLICING AND DICING IN APP PROCESSING: TARGETING PROTEASES AND MRNAS THAT REGULATE A-BETA PRODUCTION
Michael S. Wolfe, Harvard Medical School, Boston, MA, USA. Contact e-mail: [email protected] Background: Beta- and gamma-secretases catalyze production of A-beta, the major protein component of the cerebral plaques of Alzheimer’s disease, from its precursor protein (APP). While these enzymes are both aspartyl proteases and top therapeutic targets, their biochemical properties are quite different, and each presents different problems for drug discovery. Gammasecretase inhibitors should avoid blocking proteolysis of the Notch receptor, part of an essential signaling pathway in cell differentiation. Beta-secretase (BACE1) inhibitors have poor drug-like properties and do not access the brain well. OBJECTIVES: To develop Notch-sparing gamma-secretase inhibitors and to explore the regulation of mRNA splicing of BACE1 as an alternative strategy toward lowering its activity. Methods: From initial hits, iterative design, synthesis and biochemical evaluation was carried out to identify potent and selective Notch-sparing gamma-secretase inhibitors. Promising compounds were further tested in cell culture for A-beta-lowering effects and evaluated for physiochemical and pharmacokinetic properties. BACE1 alternative splicing was studied using quantitative and semi-quantitative RT-PCR methods and via a mini-gene system that recapitulates endogenous BACE1 splicing. The A-beta-generating activity of BACE1 isoforms was determined as well. Results: Iterative medicinal chemistry has led to gamma-secretase inhibitors that effectively block A-beta production and that are substantially selective with respect to Notch proteolysis. These compounds have promising physicochemical and pharmacokinetic properties, suggesting that further preclinical development is warranted. Alternative splicing of BACE1 results in isoforms with little proteolytic activity, and shunting splicing of BACE1 toward these isoforms with antisense oligonucleotides results in reduced A-beta production in cells. Cis-elements in the BACE1 pre-mRNA that regulate alternative splicing have been identified, raising the prospect of finding small molecule modulators of BACE1 splicing. Conclusions: The discovery of Notch-sparing inhibitors makes gammasecretase a much more promising therapeutic target. Beta-secretase activity can be reduced indirectly, at the level of pre-mRNA splicing, by shunting activity to inactive isoforms. S2-03-02
MOLECULAR MECHANISM OF ACTION OF GAMMA-SECRETASE INHIBITORS AND MODULATORS
Taisuke Tomita, The University of Tokyo, Tokyo, Japan. Contact e-mail: [email protected] Background: Genetic and biological studies provide strong evidence that the production and deposition of amyloid-beta peptides (Abeta) contribute to the etiology of Alzheimer’s disease (AD). Thus, gamma-secretase, that is a responsible protease for the Abeta generation, is a plausible molecular
S93
target for AD treatment. gamma-Secretase is an unusual aspartic protease that cleaves the scissile bond within the transmembrane domain. This unusual enzyme is composed of a high molecular weight membrane protein complex containing presenilin, nicastrin, Aph-1 and Pen-2. Presenilin, a catalytic subunit for the gamma-secretase, forms a ‘‘catalytic pore’’ structure in which intramembrane cleavage might be occurred. Drugs that regulate the production of Abeta by inhibiting or modulating the gamma-secretase activity could provide a disease-modifying effect on AD, although recent studies suggest that the gamma-secretase plays important roles in cellular signaling including Notch pathway. Thus, much attention is now focused on the understanding the molecular mechanism whereby the gamma-secretase inhibitors and modulators specifically regulate the Abeta-generating gamma-secretase activity. Methods: Using chemical biological technique (i.e., photoaffinity labeling), we have identified direct molecular targets of the gamma-secretase inhibitors and modulators. We have also analyzed the effects of these compounds on the catalytic pore structure using biochemical structural analysis. Results: We found that the Notch-sparing gamma-secretase inhibitor and modulator directly target the presenilin and affect the structure of the catalytic pore. Conclusions: Our results clearly indicate that presenilin is an important molecular target for the Notch-sparing gamma-secretase inhibitors as well as modulators. I will also summarize our recent studies on the gammasecretase complex, and envision the direction for developing the effective and selective gamma-secretase inhibitors as therapeutics for AD. S2-03-03
ROLE OF PRESENILIN 1 IN ADULT NEUROGENESIS
Sangram Sisodia, University of Chicago, Chicago, IL, USA. Contact e-mail: [email protected] Background: Inheritance of mutant genes encoding presenilins (PS1 and PS2) variants cause autosomal dominant, familial Alzheimer’s disease (FAD). We have investigated the influence of expressing these mutant PS1 variants on hippocampal neurogenesis in transgenic mouse models and have shown that expression of mutant PS1 leads to impairments in environmental enrichment (EE)-mediated proliferation and differentiation of hippocampal progenitor cells (NPCs). Methods: Mouse prion promoter-driven transgenes encoding either human wild-type PS1 or FAD-linked M146L and aE9 PS1 variants were exposed to standard housing conditions, or to EE, a setting that is known to induce proliferation of progenitors (NPCs) in the dentate subgranular zone and differentiation of these cells towards a neurogenic fate. In contrast to standard housing conditions, in which NPC proliferation, survival and neuronal differentiation is unaffected by expression of either wtPS1 or PS1 variants, these parameters are markedly attenuated in the hippocampus of mice expressing mutant PS1 following EE. We have shown that the proliferation and neurogenic potential of NPCs expressing either wt or mutant PS1 are indistinguishable in vitro, but that the conditioned media from microglia expressing mutant PS1 impairs the proliferation and neuronal lineage commitment of NPCs expressing wtPS1. Results: Using proteomic and high density microrray strategies, we show that the levels of secreted chemokines in the conditioned medium of microglia expressing mutant PS1 mutant, or in microglia from enriched adult mice are significantly altered and we argue that these factors are, at least in part, responsible for the defects in proliferation and neurogenesis observed in vivo. Conclusions: Current efforts are focused on the purification and molecular analysis of microglia from enriched mice and the development of a conditional transgenic mouse model that will allow us to evaluate the impact of specific cell types in the CNS on mutant PS1-mediated alterations in NPC proliferation and neurogenesis in the hippocampus. These findings support a non-cell-autonomous role for mutant PS1 in hippocampal neurogenesis. S2-03-04
REVERSE CHEMICAL GENETICS APPROACH TO IDENTIFY DRUGGABLE GENE PRODUCTS IN ALZHEIMER’S DISEASE
Tae-Wan Kim, Columbia University, New York, NY, USA. Contact e-mail: [email protected] Background: The amyloid beta-peptide (Abeta) is produced by sequential proteolytic cleavage of beta-amyloid precursor protein (APP) by a set of
S94
Focus Topics FT-02: ADNI, What’s Next? State-of-the-Art Methods for Exploring CNS Developmental and Neurodegenerative Disorders
membrane-bound proteases termed beta-secretase (BACE1) and g-secretase. The level of Abeta in the brain is critically associated with both pathological and behavioral/clinical phenotypes of AD. Thus, CNS penetrable small molecules that can reduce Abeta in an effective and safe manner remains one of the most promising approaches for the treatment of AD. While a number of direct small molecule inhibitors of beta- or g-secretase have been developed, many of them have suffered side effects owing to their target-associated toxicity. Methods: In the present study, we conducted reverse chemical genetic screening to identify small molecule inhibitors of BACE1-mediated cleavage of APP with a goal to ultimately identify a putative protein target of the identified small molecule inhibitor. In order to identify inhibitor compounds that act via a novel mechanism, we developed a cell-based assay in intact neuronal cells, based on antibody-mediated specific capture of the BACE1-derived secreted APP ectodomain (sAPPbeta) fused to a secreted alkaline phosphatase (SEAP) reporter. Results: By screening small molecule libraries using this cell system, we identified small molecules that can inhibit BACE1-mediated cleavage of APP without directly interfering with BACE1 activity. One such compound was subjected to medicinal chemistry to improve the bioactivity and the resulting compound (termed C2) reduces Abeta levels in cultured primary neurons as well as a mouse model of AD. We are currently investigating a putative cellular target for this compound. Conclusions: Identification of a small molecule modifier of the Abeta generation pathway and uncovering its cellular target may serve as the foundation for development of new therapeutic agents that could selectively probe pathological, but not normal, function of the Abeta-producing enzymes, such as BACE1. S2-03-05
IN VIVO TARGETING OF ABETA AND TAU AMYLOIDOGENESES, NEUROINFLAMMATION, CALCIUM-MEDIATED NEUROTOXICITY AND THEIR INTERPLAYS
Makoto Higuchi, National Institute of Radiological Sciences, Chiba, Japan. Contact e-mail: [email protected] Background: Pathological accumulations of amyloid beta peptide (Abeta) and tau aggregates are critically implicated in Alzheimer’s disease (AD) as initiators of the neurodegenerative cascade, while it is yet to be elucidated how these amyloidoses and downstream key processes interplay to be embodied in a self-perpetuating cycle leading to neurotoxicities. We here employed in vivo molecular imaging techniques for genetically engineered mice modeling AD to identify neurotoxic subspecies of Abeta and tau assemblies and their reciprocal links to neuroinflammation and other intermediate events in the cascade. Methods: Depositions of Abeta and tau fribrils in transgenic (Tg) mice expressing mutant amyloid precursor protein (APP) and tau were assayed by positron emission tomography (PET) with specific radioligands. Neurotoxic inflammation was visualized in these animals using PET tracers for 18-kDa translocator protein (TSPO). Results: Formation of extracellular amyloid lesions enriched with N-terminally truncated and cyclyzed Abeta, AbetaN3pE, in APP Tg mice induced amyloid PET signals characteristic of AD brains, and was enhanced by knock-down of an Abeta-degrading endopeptidase, neprilysin. Meanwhile, Abeta aggregates in hippocampal neurons undetectable by amyloid PET strongly provoked TSPO-positive microgliosis and neuronal loss. Similarly, tau fibrillogenesis prior to the emergence of PET-positive inclusions in the hippocampus of tau Tg mice gave rise to massive TSPO signals and neuronal death. This TSPO-positive neuroinflammation was accompanied by upregulation of an AbetaN3pE-producing enzyme, glutaminyl cyclase (QC), indicating interlinks among Abeta and tau depositions and toxic inflammation mediated by QC. Circumvention of excessive TSPO rises led to effective removals of Abeta and tau assemblies in immunological treatments of Tg mice with cloned microglial implants and immunizations for these molecules. Among diverse amyloid-associated events investigated here, activation of calcium-dependent proteases, calpains, was demonstrated to influence the Abeta deposition and concurrent TSPO-positive gliosis by genetically manipulating levels of a natural calpain inhibitor, calpastatin, in APP Tg mice. Conclusions: Our data illustrate crucial roles of specific proteolytic pathways in the mutual crosstalk among amyloidogen-
esis, neurotoxic glia and disrupted calcium homeostasis in animal models. Low-order multimerization of Abeta and tau may trigger neurodegenerative deteriorations, and this notion would be tested in humans with the aid of radiotracers utilized in the present study. S2-03-06
MOLECULAR DETERMINANTS OF ABETA TOXICITY
Roberto Cappai, The University of Melbourne, Melbourne, Australia. Contact e-mail: [email protected] Background: The Alzheimer’s disease Amyloid Precursor Protein is sequentially processed by beta and gamma-secretase to yield the neurotoxic Amyloid-beta (Abeta) peptide, the principal component of the senile plaques in Alzheimer’s disease brains. Abeta is a 40- 42 amino acid peptide that binds copper (Cu) and this interaction with Cu can modulate Abeta’s neurotoxic activity. The molecular basis for the Abeta and copper interaction and how it affects Abeta toxicity is being delineated. Methods: Primary mouse neuronal cortical cultures are used as the main cell based model to measure Abeta toxicity. An additional model of toxicity we use is inhibition of long term potentiation of mouse hippocampal slices. Results: The Cu:Abeta ratio modulates the Abeta folding-aggregation pathway and its neurotoxic activity. Abeta toxicity does not correlate with the ability to form amyloid, but it does did correlate with lipid peroxidation and dityrosine formation. Our sequence-activity studies, with a range of mutant peptides, have identified the histidine and tyrosine residues as being important for modulating Abeta:Cu chemistry, Abeta oligomerisation and membrane binding. We have identified phosphatidylserine as a cellular target that binds Abeta, and mediates Abeta toxicity and Abeta inhibition of long term potentiation. We are currently investigating the cell signaling pathways affected by Abeta and the role of ancillary proteins on Abeta cell binding and Abeta toxicity. Conclusions: Our current understanding of the molecular determinants of Abeta:Cu mediated toxicity is leading to the development of novel therapeutic approaches that target Abeta and inhibit either Abeta cell binding and/or Abeta toxicity. MONDAY, JULY 12, 2010 FOCUS TOPICS FT-02 ADNI, WHAT’S NEXT? STATE-OF-THE-ART METHODS FOR EXPLORING CNS DEVELOPMENTAL AND NEURODEGENERATIVE DISORDERS FT-02-01
ADNI: KEY ELEMENTS TO SUCCESS
Neil Buckholtz, National Institute on Aging, Bethesda, MD, USA. Contact e-mail: [email protected] This presentation with feature a review of the key elements that led to the successful partnerships and scientific breakthroughs made possible by ADNI. It will examine how these elements may assist in the establishment of future multi-sector efforts. FT-02-02
LEVERAGING THE PRIVATE SECTOR
Steve Paul, Lilly Research Labs, Indianapolis, IN, USA. Contact e-mail: [email protected] The significant investment from both the public and private sector was critical for the success of ADNI. This presentation will examine how similar public-private partnerships can be leveraged to expand the efforts of ADNI to focus on a broader array of neurological disorders. FT-02-03
LEVERAGING FUTURE TECHNOLOGIES
Belinda Seto, National Institute of Biomedical Imaging and Bioengineering, Bethesda, MD, USA. Contact e-mail: [email protected] Advances in imaging technologies have been critical to the success of ADNI. As efforts are undertaken to move forward and expand to other disorders of the nervous system additional technologies will be necessary. This talk will examine the future trends in technology development including neuroimaging, and how this can help shape the future efforts.