- Email: [email protected]
In vivo analysis of methylene blue and kinase inhibitors in a Tau transgenic zebrafish model
P82
Oral O1-03: Animal and Cellular Models 1
production and is elevated in Alzheimer’s disease (AD). Methods: HEK-293 cells and mouse primary neuron cultures from Tg2576 mice were analyzed for effects on BACE1 levels following glucose deprivation. Tg2576 mice were subjected to pharmacologic inhibition of energy metabolism and analyzed for BACE1 and amyloid levels. Human AD and 5XFAD mouse brain samples were analyzed in a similar fashion. Results: Energy deprivation induced phosphorylation of the translation initiation factor eIF2alpha (eIF2alpha-P), which increased the translation of BACE1. Salubrinal, an inhibitor of eIF2alpha-P phosphatase PP1c, directly increased BACE1 and elevates Abeta production in primary neurons. Preventing eIF2alpha phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominantnegative eIF2alpha kinase PERK, or PERK inhibitor P58IPK blocked the energy deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increased levels of eIF2alphaP, BACE1, Abeta, and amyloid plaques. Importantly, eIF2alpha-P and BACE1 were elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2alpha-P, and amyloid load were correlated in humans with AD. Conclusions: These results strongly suggest that eIF2alpha phosphorylation increases BACE1 levels and causes Abeta overproduction, which could be an early, initiating molecular mechanism in sporadic AD. O1-03-02
IN VIVO ANALYSIS OF METHYLENE BLUE AND KINASE INHIBITORS IN A TAU TRANSGENIC ZEBRAFISH MODEL
Christian Haass1, Dominik Paquet1, Ratan Bath2, Eva-Maria Mandelkow3, Bettina Schmid1, 1Ludwig Maimilians University, Mu¨nchen, Germany; 2 Astra Zeneca, So¨derta¨lje, Sweden; 3Max Planck Institute for Structural Molecular Biology, Hamburg, Germany. Contact e-mail: christian.haass@ med.uni-muenchen.de Background: Characteristic neuropathologic lesions of tauopathies such as Alzheimer’s disease and certain frontotemporal dementias include hyperphosphorylation and subsequent aggregation of Tau protein. Animal models for drug screening are required. Methods: We developed a novel technology for the efficient generation of transgenic zebrafish using a Gal4-UAS-based bidirectional expression system. Results: High throughput screening for inhibitors of the Tau kinase GSK3b resulted in the identification of approximately 2000 compounds. Several compounds from the pyrazine chemical series were co-crystallized with GSK3b and subsequently optimized for potency and selectivity. In tissue culture cells two novel compounds exhibited highly specific and selective inhibitory profiles for GSK3b. For in vivo validation we generated a novel Tau transgenic zebrafish model expressing the human Tau P301L mutation. Using a Gal4-UAS-based bidirectional expression system we developed a technology for the generation of highly efficient transgene expressing disease models in zebrafish, which are accessible for bio-imaging. We observed early and abnormal pathologic phosphorylation and conformation of human Tau. Early pathology allows rapid and simple screening of Tau-kinase inhibitors. While two of the newly designed GSK3b inhibitors were highly active in cultured cells, only one of them displayed in vivo activity. Moreover, a previously developed inhibitor reduced abnormal Tau phosphorylation by only 30 - 40%, while the in vivo active GSK3b inhibitor lowered abnormal Tau phosphorylation by as much as 70%. The Tau transgenic zebrafish is therefore a valuable system for efficient and rapid in vivo screening of Tau kinase inhibitors. Moreover, our new technology for zebrafish transgenesis allows modeling of numerous other human disorders, which are based on protein misfolding or overexpression. Conclusions: We developed a novel animal model for tauopathies, which allows screening and validation of drugs interfering with tau metabolisms in vivo. O1-03-03
MICROGLIA MEDIATED AND FRACTALKINE RECEPTOR DEPENDENT NEURON LOSS IN A MOUSE MODEL OF ALZHEIMER’S DISEASE
Martin Fuhrmann1, Tobias Bittner1, Christian Jung1, Steffen Burgold1, Gerda Mitteregger1, Hans Kretzschmar1, Frank M. LaFerla2, Jochen Herms1, 1 Center of Neuropathology and Prion Research, Munich, Germany; 2 Department of Neurobiology and Behavior, Irvine, CA, USA. Contact e-mail: [email protected]
Background: Microglial cells represent the immune cells of the brain. In Alzheimer’s disease they are believed to be beneficial by phagocytosing neurotoxic Abeta. Little is known about their interaction with neurons and their potential neurotoxic role in vivo.The aim was to analyze neuron-microglia interaction in Alzheimer’s disease in vivo. Additionally, the dependence of neuron-microglia interaction on the microglial fractalkine receptor (CX3CR1) was studied in homozygous triple transgenic Alzheimer’s disease mice (3xTg-AD). Methods: Long-term two-photon in vivo imaging was used to examine neuron-microglia relationship. To fluorescently label neurons and microglia, transgenic mice expressing YFP in cortical layer III and V neurons (YFP-H line) and eGFP in cerebral microglia (CX3CR1GFP/+) were crossed to homozygous 3xTg-AD mice. Hereby, quintuple transgenic mice (3xTg-AD-YFP-CX3CR1) either heterozygous (3xTgAD-YFP-CX3CR1-GFP/+) or homozygous (3xTg-AD-YFP-CX3CR1GFP/GFP) for the fractalkine receptor knock-out were yielded. Results: Selective loss of cortical layer III neurons was identified in 3xTg-ADYFP-CX3CR1-GFP/+ mice accompanied by locally increased microglial density. Complete fractalkine receptor knock-out in 3xTg-AD-YFPCX3CR1-GFP/GFP prevented this neuron loss and microglial activation. Accompanied by this, microglia exhibited a decreased surveying capacity in 3xTg-AD-YFP-CX3CR1 mice. Conclusions: Besides the useful and beneficial Abeta-phagocytosing activity of microglia in the AD brain, these cells seem to be involved in neuron loss by either directly executing neurotoxic function or by engulfing dying neurons or a combination of both. The identified neuron loss depends on a functional fractalkine receptor, which is directly involved in neuron-microglia communication. Thus, pharmacologically interfering with the fractalkine receptor may prevent or at least attenuate neuron loss in Alzheimer’s disease.
O1-03-04
TESTING THE AMYLOID CASCADE HYPOTHESIS IN A NOVEL MOUSE MODEL OF ALZHEIMER’S DISEASE THAT SHOWS COMPLETE DISEASE PROGRESSION
Carol A. Colton1, Michael P. Vitek2, Donna Wilcock1, 1Duke University Medical School, Durham, NC, USA; 2Cognosci, Inc., Research Triangle Park, NC, USA. Contact e-mail: [email protected] Background: The amyloid hypothesis of Alzheimer’s disease states that Abeta production and deposition lead in sequential fashion to tau hyperphosphorylation and aggregation forming neurofibrillary tangles which then leads, in turn, to neuronal loss and cognitive decline. If true, then reducing levels of amyloid to stop downstream pathologies is a logical and potentially viable therapeutic intervention. To date, animal studies using an immunotherapeutic approach to reduce brain amyloid have only examined restricted changes in AD pathology such as amyloid deposition or tau pathology composed of mutant human tau that is not truly representative of AD. While providing useful information, these limited models have lacked neuronal loss. Thus, it is unclear if targeting amyloid is sufficient to reduce pathological, non-mutated tau and neuronal loss. Methods: We have developed 2 transgenic mouse strains (APPSw/NOS2-/- and APPSwDI/NOS2-/-) that more closely model Alzheimer’s disease. By combining APP with NOS2 disruption (Nitric Oxide Synthase 2 knockout) these mice show amyloid pathology, hyperphosphorylated and aggregated normal mouse tau, significant neuron loss and cognitive deficits. NOS2-/- mice alone do not develop AD-like pathology. Using active immunization, the effects of lowering Abeta on mouse tau pathology, neuron loss and cognition in our novel mouse models was
Oral O1-03: Animal and Cellular Models 1
production and is elevated in Alzheimer’s disease (AD). Methods: HEK-293 cells and mouse primary neuron cultures from Tg2576 mice were analyzed for effects on BACE1 levels following glucose deprivation. Tg2576 mice were subjected to pharmacologic inhibition of energy metabolism and analyzed for BACE1 and amyloid levels. Human AD and 5XFAD mouse brain samples were analyzed in a similar fashion. Results: Energy deprivation induced phosphorylation of the translation initiation factor eIF2alpha (eIF2alpha-P), which increased the translation of BACE1. Salubrinal, an inhibitor of eIF2alpha-P phosphatase PP1c, directly increased BACE1 and elevates Abeta production in primary neurons. Preventing eIF2alpha phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominantnegative eIF2alpha kinase PERK, or PERK inhibitor P58IPK blocked the energy deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increased levels of eIF2alphaP, BACE1, Abeta, and amyloid plaques. Importantly, eIF2alpha-P and BACE1 were elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2alpha-P, and amyloid load were correlated in humans with AD. Conclusions: These results strongly suggest that eIF2alpha phosphorylation increases BACE1 levels and causes Abeta overproduction, which could be an early, initiating molecular mechanism in sporadic AD. O1-03-02
IN VIVO ANALYSIS OF METHYLENE BLUE AND KINASE INHIBITORS IN A TAU TRANSGENIC ZEBRAFISH MODEL
Christian Haass1, Dominik Paquet1, Ratan Bath2, Eva-Maria Mandelkow3, Bettina Schmid1, 1Ludwig Maimilians University, Mu¨nchen, Germany; 2 Astra Zeneca, So¨derta¨lje, Sweden; 3Max Planck Institute for Structural Molecular Biology, Hamburg, Germany. Contact e-mail: christian.haass@ med.uni-muenchen.de Background: Characteristic neuropathologic lesions of tauopathies such as Alzheimer’s disease and certain frontotemporal dementias include hyperphosphorylation and subsequent aggregation of Tau protein. Animal models for drug screening are required. Methods: We developed a novel technology for the efficient generation of transgenic zebrafish using a Gal4-UAS-based bidirectional expression system. Results: High throughput screening for inhibitors of the Tau kinase GSK3b resulted in the identification of approximately 2000 compounds. Several compounds from the pyrazine chemical series were co-crystallized with GSK3b and subsequently optimized for potency and selectivity. In tissue culture cells two novel compounds exhibited highly specific and selective inhibitory profiles for GSK3b. For in vivo validation we generated a novel Tau transgenic zebrafish model expressing the human Tau P301L mutation. Using a Gal4-UAS-based bidirectional expression system we developed a technology for the generation of highly efficient transgene expressing disease models in zebrafish, which are accessible for bio-imaging. We observed early and abnormal pathologic phosphorylation and conformation of human Tau. Early pathology allows rapid and simple screening of Tau-kinase inhibitors. While two of the newly designed GSK3b inhibitors were highly active in cultured cells, only one of them displayed in vivo activity. Moreover, a previously developed inhibitor reduced abnormal Tau phosphorylation by only 30 - 40%, while the in vivo active GSK3b inhibitor lowered abnormal Tau phosphorylation by as much as 70%. The Tau transgenic zebrafish is therefore a valuable system for efficient and rapid in vivo screening of Tau kinase inhibitors. Moreover, our new technology for zebrafish transgenesis allows modeling of numerous other human disorders, which are based on protein misfolding or overexpression. Conclusions: We developed a novel animal model for tauopathies, which allows screening and validation of drugs interfering with tau metabolisms in vivo. O1-03-03
MICROGLIA MEDIATED AND FRACTALKINE RECEPTOR DEPENDENT NEURON LOSS IN A MOUSE MODEL OF ALZHEIMER’S DISEASE
Martin Fuhrmann1, Tobias Bittner1, Christian Jung1, Steffen Burgold1, Gerda Mitteregger1, Hans Kretzschmar1, Frank M. LaFerla2, Jochen Herms1, 1 Center of Neuropathology and Prion Research, Munich, Germany; 2 Department of Neurobiology and Behavior, Irvine, CA, USA. Contact e-mail: [email protected]
Background: Microglial cells represent the immune cells of the brain. In Alzheimer’s disease they are believed to be beneficial by phagocytosing neurotoxic Abeta. Little is known about their interaction with neurons and their potential neurotoxic role in vivo.The aim was to analyze neuron-microglia interaction in Alzheimer’s disease in vivo. Additionally, the dependence of neuron-microglia interaction on the microglial fractalkine receptor (CX3CR1) was studied in homozygous triple transgenic Alzheimer’s disease mice (3xTg-AD). Methods: Long-term two-photon in vivo imaging was used to examine neuron-microglia relationship. To fluorescently label neurons and microglia, transgenic mice expressing YFP in cortical layer III and V neurons (YFP-H line) and eGFP in cerebral microglia (CX3CR1GFP/+) were crossed to homozygous 3xTg-AD mice. Hereby, quintuple transgenic mice (3xTg-AD-YFP-CX3CR1) either heterozygous (3xTgAD-YFP-CX3CR1-GFP/+) or homozygous (3xTg-AD-YFP-CX3CR1GFP/GFP) for the fractalkine receptor knock-out were yielded. Results: Selective loss of cortical layer III neurons was identified in 3xTg-ADYFP-CX3CR1-GFP/+ mice accompanied by locally increased microglial density. Complete fractalkine receptor knock-out in 3xTg-AD-YFPCX3CR1-GFP/GFP prevented this neuron loss and microglial activation. Accompanied by this, microglia exhibited a decreased surveying capacity in 3xTg-AD-YFP-CX3CR1 mice. Conclusions: Besides the useful and beneficial Abeta-phagocytosing activity of microglia in the AD brain, these cells seem to be involved in neuron loss by either directly executing neurotoxic function or by engulfing dying neurons or a combination of both. The identified neuron loss depends on a functional fractalkine receptor, which is directly involved in neuron-microglia communication. Thus, pharmacologically interfering with the fractalkine receptor may prevent or at least attenuate neuron loss in Alzheimer’s disease.
O1-03-04
TESTING THE AMYLOID CASCADE HYPOTHESIS IN A NOVEL MOUSE MODEL OF ALZHEIMER’S DISEASE THAT SHOWS COMPLETE DISEASE PROGRESSION
Carol A. Colton1, Michael P. Vitek2, Donna Wilcock1, 1Duke University Medical School, Durham, NC, USA; 2Cognosci, Inc., Research Triangle Park, NC, USA. Contact e-mail: [email protected] Background: The amyloid hypothesis of Alzheimer’s disease states that Abeta production and deposition lead in sequential fashion to tau hyperphosphorylation and aggregation forming neurofibrillary tangles which then leads, in turn, to neuronal loss and cognitive decline. If true, then reducing levels of amyloid to stop downstream pathologies is a logical and potentially viable therapeutic intervention. To date, animal studies using an immunotherapeutic approach to reduce brain amyloid have only examined restricted changes in AD pathology such as amyloid deposition or tau pathology composed of mutant human tau that is not truly representative of AD. While providing useful information, these limited models have lacked neuronal loss. Thus, it is unclear if targeting amyloid is sufficient to reduce pathological, non-mutated tau and neuronal loss. Methods: We have developed 2 transgenic mouse strains (APPSw/NOS2-/- and APPSwDI/NOS2-/-) that more closely model Alzheimer’s disease. By combining APP with NOS2 disruption (Nitric Oxide Synthase 2 knockout) these mice show amyloid pathology, hyperphosphorylated and aggregated normal mouse tau, significant neuron loss and cognitive deficits. NOS2-/- mice alone do not develop AD-like pathology. Using active immunization, the effects of lowering Abeta on mouse tau pathology, neuron loss and cognition in our novel mouse models was