- Email: [email protected]
P1-118
S130
Poster Presentations P1
be observed from 2.5M of age. Doxycycline suppresses transgene expression, reduces abnormal tau pathology and improves cognitive performance. Objective: The aim of this study was to determine how quickly doxycycline treatment suppressed the transgene expression, reduced transgenic protein levels and altered abnormal tau pathology. Methods: rTg4510 mice were dosed with doxycycline or control diet for 1, 6 or 12 weeks. The transgene expression levels were then measured by in situ hybridization, the soluble and insoluble tau protein levels were determined by Western analysis and the neurofibrillary pathology was examined by immunohistochemistry using standard techniques. Results: Consistent with our previous findings, tau pathology is progressive with initial neuropil staining followed by pre-tangle and tangle pathology. Maximal transgene suppression (67-80%) occurred after only 1 week of dox dosing. However after 1 week of doxycycline dosing there were no marked changes in tau pathology; tau neuropil staining and occasional pretangles were present in the cortex and hippocampus. Following 6 or 12 weeks doxycycline administration, the tau neuropil immunoreactivity was markedly reduced throughout the forebrain and although the neurofibrillary pathology was significantly decreased in rTg4510 mice compared to control mice, the number of pre-tangles and tangles increased with age. Conclusions: Maximal suppression of transgenic tau expression occurs within 1 week of transgenic tau repression; however, this did not allow sufficient time to reduce tau pathology. Clearance of tau pathology occurs in the neuropil after 6 and 12 weeks of tau repression. Pre-tangle pathology develops at a slower rate, but pre-tangles continue to convert to dense, mature tangles with tau repression. Despite this, neurodegeneration appears to slow or halt. P1-118
ESTABLISHMENT OF BRAIN SLICE CULTURES FROM ALPHA SYNUCLEIN TRANSGENIC MICE FOR DRUG SCREENING
Robert Wronski1, Birgit Hutter-Paier1, Michael Schlossmacher2, Valerie Cullen2, Evelyn Schauer1, Manfred Windisch1. 1JSW-Research, Graz, Austria; 2Brigham & Women’s Hospital, Boston, MA, USA. Contact e-mail: [email protected] Background: Lewy bodies are associated with Parkinson’s (PD) and Alzheimer’s diseases (AD) and so far it is not understood whether dementia with Lewy bodies is a distinct clinical entity or a variant of AD or PD. Recent research has revealed that Lewy bodies contain deposits of alphasynuclein, a protein linked to PD and multiple system atrophy. Therefore transgenic mice over-expressing human alpha-synuclein are suitable models to study the influence of drugs on these brain depositions and on alpha-synuclein production, sequestration and deposition. Objective(s): For the present study mice over-expressing human alpha-synuclein under the control of the neuron-specific PDGF promoter and their littermates were used. Alpha-synuclein is expressed in high levels, resulting in an age-dependent increase of aggregated alpha-synuclein and Lewy body like inclusions, a pathological relevant situation for several diseases. Methods: Brain slice cultures were prepared from transgenic alpha-synuclein mice of different age for establishing a range of alpha-synuclein content in neural tissue. Therefore multiple organotypic brain slice experiments were performed on postnatal day 7 mouse pups. 300M brain slices containing the cortex and the striatum were collected and transferred to the culture chamber membrane resting in ice cold slicing media. For each postnatal day 7 pup, approximately four slices were collected and placed at 4°C for two hours to allow the cells to recover and then transferred to the incubator overnight. Plating media were removed every 24, 48 to 72 hours and replaced with fresh media until the exact culture period of 1, 4, 7, 10 and 13 days was reached. At this point medium and slices were sampled and alpha-synuclein content was determined with ELISA. Some slices were further immunohistochemically evaluated using antibodies against human alpha-synuclein, ubiquitin and tyrosine hydroxylase. Results: Although evaluation is currently under progress staining for alpha-synuclein and ubiquitin reveals a constant increase of this protein in slice cultures. Conclusions: Before going into in vivo studies such brain slice cultures would represent an excellent in vitro tool to test new compounds which
might be relevant for the treatment of disorders like PD and AD which as a central pathological hallmark exhibit formation of protein aggregation. P1-119
ESTABLISHMENT OF A P75NTR TRANSGENIC MOUSE MODEL
Gert Schwach1, Ulrike Bauer1, Birgit Hutter-Paier1, Manuela Prokesch1, Gerhard Kostner2, Antonello D’Arrigo3, Manfred Windisch1. 1JSWResearch, Graz, Austria; 2Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria; 3Research and Innovation (R&I), Padova, Italy. Contact e-mail: [email protected] Background: p75 neurotrophin receptor (p75NTR) is a type I transmembrane receptor structurally related to the tumor necrosis factor receptor superfamily of death receptors. p75NTR binds non-neurotrophin ligands, including amyloid beta peptides (Abeta), known to abnormally accumulate in brains of Alzheimer Disease (AD) patients, and pro-NGF (Nerve Growth Factor) which is shown to be upregulated in AD brains. The abnormal accumulation of Abeta in the form of senile plaques is one of the main characteristics of AD. It has been shown that two members of the neurotrophin receptor superfamily, TrkA (tyrosine kinase receptor A) and p75NTR differentially regulate the processing of APP (amyloid precursor protein). TrkA reduces, whereas p75NTR activates the beta-cleavage of APP. The p75NTR-dependent effect requires NGF binding and activation of the second messenger ceramide. In addition normal aging activates Abeta generation in the brain by ‘switching’ from the TrkA to the p75NTR receptor system. Objective(s): In our study we have generated several hp75NTR transgenic mouse lines under control of the murine Thy-1promoter in order to induce neuronal overexpression of this receptor. Methods: Three different mouse lines were intensively investigated. p75 expression, behavioral changes due to the transgene expression as well as brain histomorphological changes were evaluated. Results: p75NTR mRNA and protein expression was detected in different brain regions as well as in spinal cord of tg mice. The Morris-Water-Maze test showed significant cognition disturbance in 8 month old transgenic mice. For the same groups of mice no difference could be detected between transgenic mice and non-transgenic littermates in the Open Field and in the RotaRod tests, indicating normal psychomotoric activity and motor function. Conclusions: A mouse line overexpressing full length hp75NTR could be a useful experimental animal model for evaluating the role of hp75NTR in neurodegenerative processes and its effects on human Abeta generation or its relationship with other ligands in vivo. P1-120
SMALL ARCTIC A AGGREGATES DISTURB SYNAPTIC PLASTICITY AND BEHAVIOR IN TRANSGENIC MICE
Marlen Knobloch1, Melissa Farinelli2, Uwe Konietzko1, Isabelle M. Mansuy2, Roger M. Nitsch1. 1Division of Psychiatry Research, University of Zurich, Switzerland; 2Brain Research Institute, University of Zurich, Switzerland. Contact e-mail: [email protected] Background: Although it is widely accepted that A plays an important role in Alzheimer’s disease (AD), the underlying pathogenic mechanisms are still unclear. Recently, smaller oligomeric and protofibrillar A aggregates have come into focus and several experiments demonstrated a highly neurotoxic effect of these species in vitro. The discovery of the Arctic mutation, a familial AD point mutation within the A sequence of APP leading to the formation of more protofibrils, underscored a role of small A oligomers in AD. Objectives: We have generated a new transgenic mouse model overexpressing human APP with the Swedish and Arctic mutations. With combined expression of these two mutations in a single construct we expected an increased A production followed by accelerated aggregation and protofibril formation, providing an in vivo tool to further elucidate the role of small A aggregates in AD. Results: Swe/Arc-APP transgenic mice showed severe cognitive impairments already at the age of 6 to 7 months in the Y-maze, the Morris Water maze and the Two-way
Poster Presentations P1
be observed from 2.5M of age. Doxycycline suppresses transgene expression, reduces abnormal tau pathology and improves cognitive performance. Objective: The aim of this study was to determine how quickly doxycycline treatment suppressed the transgene expression, reduced transgenic protein levels and altered abnormal tau pathology. Methods: rTg4510 mice were dosed with doxycycline or control diet for 1, 6 or 12 weeks. The transgene expression levels were then measured by in situ hybridization, the soluble and insoluble tau protein levels were determined by Western analysis and the neurofibrillary pathology was examined by immunohistochemistry using standard techniques. Results: Consistent with our previous findings, tau pathology is progressive with initial neuropil staining followed by pre-tangle and tangle pathology. Maximal transgene suppression (67-80%) occurred after only 1 week of dox dosing. However after 1 week of doxycycline dosing there were no marked changes in tau pathology; tau neuropil staining and occasional pretangles were present in the cortex and hippocampus. Following 6 or 12 weeks doxycycline administration, the tau neuropil immunoreactivity was markedly reduced throughout the forebrain and although the neurofibrillary pathology was significantly decreased in rTg4510 mice compared to control mice, the number of pre-tangles and tangles increased with age. Conclusions: Maximal suppression of transgenic tau expression occurs within 1 week of transgenic tau repression; however, this did not allow sufficient time to reduce tau pathology. Clearance of tau pathology occurs in the neuropil after 6 and 12 weeks of tau repression. Pre-tangle pathology develops at a slower rate, but pre-tangles continue to convert to dense, mature tangles with tau repression. Despite this, neurodegeneration appears to slow or halt. P1-118
ESTABLISHMENT OF BRAIN SLICE CULTURES FROM ALPHA SYNUCLEIN TRANSGENIC MICE FOR DRUG SCREENING
Robert Wronski1, Birgit Hutter-Paier1, Michael Schlossmacher2, Valerie Cullen2, Evelyn Schauer1, Manfred Windisch1. 1JSW-Research, Graz, Austria; 2Brigham & Women’s Hospital, Boston, MA, USA. Contact e-mail: [email protected] Background: Lewy bodies are associated with Parkinson’s (PD) and Alzheimer’s diseases (AD) and so far it is not understood whether dementia with Lewy bodies is a distinct clinical entity or a variant of AD or PD. Recent research has revealed that Lewy bodies contain deposits of alphasynuclein, a protein linked to PD and multiple system atrophy. Therefore transgenic mice over-expressing human alpha-synuclein are suitable models to study the influence of drugs on these brain depositions and on alpha-synuclein production, sequestration and deposition. Objective(s): For the present study mice over-expressing human alpha-synuclein under the control of the neuron-specific PDGF promoter and their littermates were used. Alpha-synuclein is expressed in high levels, resulting in an age-dependent increase of aggregated alpha-synuclein and Lewy body like inclusions, a pathological relevant situation for several diseases. Methods: Brain slice cultures were prepared from transgenic alpha-synuclein mice of different age for establishing a range of alpha-synuclein content in neural tissue. Therefore multiple organotypic brain slice experiments were performed on postnatal day 7 mouse pups. 300M brain slices containing the cortex and the striatum were collected and transferred to the culture chamber membrane resting in ice cold slicing media. For each postnatal day 7 pup, approximately four slices were collected and placed at 4°C for two hours to allow the cells to recover and then transferred to the incubator overnight. Plating media were removed every 24, 48 to 72 hours and replaced with fresh media until the exact culture period of 1, 4, 7, 10 and 13 days was reached. At this point medium and slices were sampled and alpha-synuclein content was determined with ELISA. Some slices were further immunohistochemically evaluated using antibodies against human alpha-synuclein, ubiquitin and tyrosine hydroxylase. Results: Although evaluation is currently under progress staining for alpha-synuclein and ubiquitin reveals a constant increase of this protein in slice cultures. Conclusions: Before going into in vivo studies such brain slice cultures would represent an excellent in vitro tool to test new compounds which
might be relevant for the treatment of disorders like PD and AD which as a central pathological hallmark exhibit formation of protein aggregation. P1-119
ESTABLISHMENT OF A P75NTR TRANSGENIC MOUSE MODEL
Gert Schwach1, Ulrike Bauer1, Birgit Hutter-Paier1, Manuela Prokesch1, Gerhard Kostner2, Antonello D’Arrigo3, Manfred Windisch1. 1JSWResearch, Graz, Austria; 2Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria; 3Research and Innovation (R&I), Padova, Italy. Contact e-mail: [email protected] Background: p75 neurotrophin receptor (p75NTR) is a type I transmembrane receptor structurally related to the tumor necrosis factor receptor superfamily of death receptors. p75NTR binds non-neurotrophin ligands, including amyloid beta peptides (Abeta), known to abnormally accumulate in brains of Alzheimer Disease (AD) patients, and pro-NGF (Nerve Growth Factor) which is shown to be upregulated in AD brains. The abnormal accumulation of Abeta in the form of senile plaques is one of the main characteristics of AD. It has been shown that two members of the neurotrophin receptor superfamily, TrkA (tyrosine kinase receptor A) and p75NTR differentially regulate the processing of APP (amyloid precursor protein). TrkA reduces, whereas p75NTR activates the beta-cleavage of APP. The p75NTR-dependent effect requires NGF binding and activation of the second messenger ceramide. In addition normal aging activates Abeta generation in the brain by ‘switching’ from the TrkA to the p75NTR receptor system. Objective(s): In our study we have generated several hp75NTR transgenic mouse lines under control of the murine Thy-1promoter in order to induce neuronal overexpression of this receptor. Methods: Three different mouse lines were intensively investigated. p75 expression, behavioral changes due to the transgene expression as well as brain histomorphological changes were evaluated. Results: p75NTR mRNA and protein expression was detected in different brain regions as well as in spinal cord of tg mice. The Morris-Water-Maze test showed significant cognition disturbance in 8 month old transgenic mice. For the same groups of mice no difference could be detected between transgenic mice and non-transgenic littermates in the Open Field and in the RotaRod tests, indicating normal psychomotoric activity and motor function. Conclusions: A mouse line overexpressing full length hp75NTR could be a useful experimental animal model for evaluating the role of hp75NTR in neurodegenerative processes and its effects on human Abeta generation or its relationship with other ligands in vivo. P1-120
SMALL ARCTIC A AGGREGATES DISTURB SYNAPTIC PLASTICITY AND BEHAVIOR IN TRANSGENIC MICE
Marlen Knobloch1, Melissa Farinelli2, Uwe Konietzko1, Isabelle M. Mansuy2, Roger M. Nitsch1. 1Division of Psychiatry Research, University of Zurich, Switzerland; 2Brain Research Institute, University of Zurich, Switzerland. Contact e-mail: [email protected] Background: Although it is widely accepted that A plays an important role in Alzheimer’s disease (AD), the underlying pathogenic mechanisms are still unclear. Recently, smaller oligomeric and protofibrillar A aggregates have come into focus and several experiments demonstrated a highly neurotoxic effect of these species in vitro. The discovery of the Arctic mutation, a familial AD point mutation within the A sequence of APP leading to the formation of more protofibrils, underscored a role of small A oligomers in AD. Objectives: We have generated a new transgenic mouse model overexpressing human APP with the Swedish and Arctic mutations. With combined expression of these two mutations in a single construct we expected an increased A production followed by accelerated aggregation and protofibril formation, providing an in vivo tool to further elucidate the role of small A aggregates in AD. Results: Swe/Arc-APP transgenic mice showed severe cognitive impairments already at the age of 6 to 7 months in the Y-maze, the Morris Water maze and the Two-way