- Email: [email protected]
P2-110 Amyloid-induced neurofibrillary tangle formation addressed in vivo and in vitro
Poster Session P2: Animal and Cellular Models - Animal Models, Transgenic AD phases. Between 6 and 30 months all phases were found in APP23, while APP24 (lower expression) and APP51 only reach phase 4 and 3, respectively. Crossbreeding with PS mice accelerated deposition to phase 5. Diffuse, compact and vascular amyloid was found in all lines with age and region dependent differences in the proportion of the various forms. Compact plaques (bigger and more dense than in AD) and vascular amyloid was most pronounced in APP23, whereas APP24 and APP51 showed primarily diffuse and less compact or vascular A[3 deposits. Cored plaques were relatively more abundant in APP51 than in both other lines. These differences remained largely unchanged after crossbreeding with PS mice. Conclusions: A[3 deposition in all APP transgenic mice analyzed follows a similar regional hierarchy as seen in AD. Although the different forms of amyloid can be found in all lines there is a considerable variation in abundance. This seems to dependent on the APP expression level and APP mutation used. Different APP transgenic mouse lines are required to study the various aspects of amyloidosis in AD.
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DROSOPHILA MODELS OF ALZHEIMERS-RELATED PATHWAYS
Mary Konsolaki*, Ho-Juhn Song, Wesley Dobbs, Dan Garza. Novartis Institutes of Biomedical Research, Cambridge, MA, USA. Contact e-mail: mary. [email protected], corn
Background: Model organisms such as Drosophila melanogaster are increasingly used for in vivo studies of mechanisms of neurodegeneration, including Atzheimer's disease. Flies can closely mimic phenotypic, behavioral and physiological aspects of Alzheimer's disease and can be used for in vivo validation as well as elucidation of novel disease pathways. Objective(s): We will present our Drosophila melanogaster models of Alzheimer's-related pathways. These models address mechanisms of Abeta-mediated toxicity and gamma-secretase processing. Results: We have expressed human Abeta peptides in the CNS of flies and show that such expression causes visible and behavioral phenotypes that are manifested in an age dependent manner. We also show that Abeta peptides are secreted from fly cells and accumulate in fly tissues over age. Taking advantage of well established Drosophila tools, we have conducted genetic screens aimed at the identification of genes that modify Abeta-associated phenotypes. One such gene was found to be neprilysin, a metalloprotease known to degrade Abeta. In our in vivo system, we show that modest upregulation of the endogenous nepfilysin activity is sufficient for suppression of the Abeta-associated phenotypes and reduction of Abeta levels. In a complementary model, we have expressed the APP-C99 pre-processed form of the amyloid precursor and shown that such expression also causes distinct phenotypes. We will present our efforts at identifying factors that act in the C99-gamma-secretase pathway, through genetic screens. Conclusions: Our Drosophila models can serve as tools for the understanding of mechanisms of Abeta toxicity as well as gamma-secretase processing, through genetic analyses.
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AMYLOID-INDUCED NEUROFIBRILLARY TANGLE FORMATION ADDRESSED IN VIVO AND IN VITRO
Luis Pennanen, Della David, Frederic Hoerndli, Pascal Kurosinski, Feng Chen, Andreas Schild, Alessandra Ferrari, Roger M. Nitsch, Jiirgen G6tz*, University of Zi,irich, Zurich, Switzerland. Contact e-mail: goetz @bli.unizh.ch Tan pathology characterizes a number of neurodegenerative disorders including frontotemporal dementia (FTD) and Alzheimer's disease (AD). In each disease the microtubule-associated protein tau is hyperphosphorylated and in a filamentous form. The neurofihrillar pathology can be reproduced by transgenic expression of tan mutations associated with the inherited dementia FTDP-17, that has similarities to AD. Transgenic mice expressing the FTDP-17 mutation P301L of tan recapitulate key features of the human pathology, i.e., tau proteins aggregate and neurofihrillary tangles (NFI') begin to appear in the amygdala at six months of age. To detect early signs of tau aggregate-associated changes, we investigated behavioral alterations and cognitive deficits in these mice using an amygdala-specific test battery
$255
for anxiety-related and cognitive behavior. P301L mice had anxiety levels not different from wild-types, but their exploratory behavior was significantly increased. Acquisition of a fear response to tone and context as well as taste aversion was comparable to wild-types. However, extinction of a conditioned taste aversion was significantly accelerated. We conclude that already the aggregation of tau proteins not yet accompanied by massive formation of NFT causes selective behavioral deficits. To gain insight into pathogenic mechanisms, we initiated a transcriptomic analysis of the P301L mice and identified several differentially expressed genes including one encoding an enzyme involved in the detoxification of cells. Analysis of AD brain sections revealed accumulation of this enzyme in NFT-bearing neurons. A proteomic analysis of differentially extracted brains using 2-dimensional gel electrophoresis and mass spectrometry identified 19 up- and 27 downregulated proteins. Beta-amyloid-induced NFT formation in the P301L mice has been shown to be associated with the phosphorylation of tan at the $422 and the AT100 epitope. Therefore, we established a human tissue-culture system of beta-amyloid-mediated tan filament formation. Mutagenesis of the $422 epitope of tau abrogated the beta-amyloid-mediated decrease in tan solubility and tan filament formation. Additional mutants have been generated to determine the role of tan phosphorylation sites in addition to $422 in tau aggregation.
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STAGING OF NEUROPATHOLOGICAL CHANGES IN A X O N ' S N O V E L T R A N S G E N I C AD RAT M O D E L IS L I N K E D T O A L E T H A L P H E N O T Y P E
Norbert Zilka* 1, Natalia Csokova i, Lubica Vechterova 1, Michaela Skrabanoval, Miroslava Hrnkova 1, Peter Filipcik 2, Michal Novak 1'2. 1Institute of Neuroimmunology, Bratislava, Slovakia; 2Axon Neuroscience, Vienna, Austria. Contact e-mail: [email protected] Background: In Alzheimer's disease the load of neurofibrillary tangles (NFT) correlates well with the clinical manifestations of the disease. The major constituent of NFT is tan protein of which the majority present in human AD brain is truncated. A "functionally active" subpopulation of these truncated forms - designated Alzheimer's truncated tan - leads to development of neurofibrillary degeneration when expressed as transgene in the rat brain. Objective(s): The aim of the present work was to characterize our new transgenic rat AD model at the histological, immunohistochemical and biochemical level. Methods: For detection of NFTs, neuropil threads and dystrophic neurites, we have used Gallyas silver staining method, Bielschowski silver technique, staining with histochemical dyes that bind crossed beta-pleated sheet structures (Thiottavin S, Congo Red) and immunohistochemical labelling using phosphorylation and conformation dependent anti tan antibodies. At the biochemical level, a series of ultracentrifugation and sarkosyl extraction protocols was used to obtain soluble and insoluble tan fractions. Results: Expression of Alzheimer's truncated tau in transgehic rat AD model results in formation of intracellular pretangles in the cortex (fig. A), hippocampus, striatum and brain stem.
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DROSOPHILA MODELS OF ALZHEIMERS-RELATED PATHWAYS
Mary Konsolaki*, Ho-Juhn Song, Wesley Dobbs, Dan Garza. Novartis Institutes of Biomedical Research, Cambridge, MA, USA. Contact e-mail: mary. [email protected], corn
Background: Model organisms such as Drosophila melanogaster are increasingly used for in vivo studies of mechanisms of neurodegeneration, including Atzheimer's disease. Flies can closely mimic phenotypic, behavioral and physiological aspects of Alzheimer's disease and can be used for in vivo validation as well as elucidation of novel disease pathways. Objective(s): We will present our Drosophila melanogaster models of Alzheimer's-related pathways. These models address mechanisms of Abeta-mediated toxicity and gamma-secretase processing. Results: We have expressed human Abeta peptides in the CNS of flies and show that such expression causes visible and behavioral phenotypes that are manifested in an age dependent manner. We also show that Abeta peptides are secreted from fly cells and accumulate in fly tissues over age. Taking advantage of well established Drosophila tools, we have conducted genetic screens aimed at the identification of genes that modify Abeta-associated phenotypes. One such gene was found to be neprilysin, a metalloprotease known to degrade Abeta. In our in vivo system, we show that modest upregulation of the endogenous nepfilysin activity is sufficient for suppression of the Abeta-associated phenotypes and reduction of Abeta levels. In a complementary model, we have expressed the APP-C99 pre-processed form of the amyloid precursor and shown that such expression also causes distinct phenotypes. We will present our efforts at identifying factors that act in the C99-gamma-secretase pathway, through genetic screens. Conclusions: Our Drosophila models can serve as tools for the understanding of mechanisms of Abeta toxicity as well as gamma-secretase processing, through genetic analyses.
~
AMYLOID-INDUCED NEUROFIBRILLARY TANGLE FORMATION ADDRESSED IN VIVO AND IN VITRO
Luis Pennanen, Della David, Frederic Hoerndli, Pascal Kurosinski, Feng Chen, Andreas Schild, Alessandra Ferrari, Roger M. Nitsch, Jiirgen G6tz*, University of Zi,irich, Zurich, Switzerland. Contact e-mail: goetz @bli.unizh.ch Tan pathology characterizes a number of neurodegenerative disorders including frontotemporal dementia (FTD) and Alzheimer's disease (AD). In each disease the microtubule-associated protein tau is hyperphosphorylated and in a filamentous form. The neurofihrillar pathology can be reproduced by transgenic expression of tan mutations associated with the inherited dementia FTDP-17, that has similarities to AD. Transgenic mice expressing the FTDP-17 mutation P301L of tan recapitulate key features of the human pathology, i.e., tau proteins aggregate and neurofihrillary tangles (NFI') begin to appear in the amygdala at six months of age. To detect early signs of tau aggregate-associated changes, we investigated behavioral alterations and cognitive deficits in these mice using an amygdala-specific test battery
$255
for anxiety-related and cognitive behavior. P301L mice had anxiety levels not different from wild-types, but their exploratory behavior was significantly increased. Acquisition of a fear response to tone and context as well as taste aversion was comparable to wild-types. However, extinction of a conditioned taste aversion was significantly accelerated. We conclude that already the aggregation of tau proteins not yet accompanied by massive formation of NFT causes selective behavioral deficits. To gain insight into pathogenic mechanisms, we initiated a transcriptomic analysis of the P301L mice and identified several differentially expressed genes including one encoding an enzyme involved in the detoxification of cells. Analysis of AD brain sections revealed accumulation of this enzyme in NFT-bearing neurons. A proteomic analysis of differentially extracted brains using 2-dimensional gel electrophoresis and mass spectrometry identified 19 up- and 27 downregulated proteins. Beta-amyloid-induced NFT formation in the P301L mice has been shown to be associated with the phosphorylation of tan at the $422 and the AT100 epitope. Therefore, we established a human tissue-culture system of beta-amyloid-mediated tan filament formation. Mutagenesis of the $422 epitope of tau abrogated the beta-amyloid-mediated decrease in tan solubility and tan filament formation. Additional mutants have been generated to determine the role of tan phosphorylation sites in addition to $422 in tau aggregation.
F
P
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2
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STAGING OF NEUROPATHOLOGICAL CHANGES IN A X O N ' S N O V E L T R A N S G E N I C AD RAT M O D E L IS L I N K E D T O A L E T H A L P H E N O T Y P E
Norbert Zilka* 1, Natalia Csokova i, Lubica Vechterova 1, Michaela Skrabanoval, Miroslava Hrnkova 1, Peter Filipcik 2, Michal Novak 1'2. 1Institute of Neuroimmunology, Bratislava, Slovakia; 2Axon Neuroscience, Vienna, Austria. Contact e-mail: [email protected] Background: In Alzheimer's disease the load of neurofibrillary tangles (NFT) correlates well with the clinical manifestations of the disease. The major constituent of NFT is tan protein of which the majority present in human AD brain is truncated. A "functionally active" subpopulation of these truncated forms - designated Alzheimer's truncated tan - leads to development of neurofibrillary degeneration when expressed as transgene in the rat brain. Objective(s): The aim of the present work was to characterize our new transgenic rat AD model at the histological, immunohistochemical and biochemical level. Methods: For detection of NFTs, neuropil threads and dystrophic neurites, we have used Gallyas silver staining method, Bielschowski silver technique, staining with histochemical dyes that bind crossed beta-pleated sheet structures (Thiottavin S, Congo Red) and immunohistochemical labelling using phosphorylation and conformation dependent anti tan antibodies. At the biochemical level, a series of ultracentrifugation and sarkosyl extraction protocols was used to obtain soluble and insoluble tan fractions. Results: Expression of Alzheimer's truncated tau in transgehic rat AD model results in formation of intracellular pretangles in the cortex (fig. A), hippocampus, striatum and brain stem.