- Email: [email protected]
P3-257 Search for small molecule inhibitors of tau aggregation into Alzheimer paired helical filaments
S428
~5-7]
Poster Session P3: Molecular Mechanisms of Neurodegeneration - Neurofibrillary Degeneration/Tau
SEARCH F O R S M A L L M O L E C U L E INHIBITORS OF
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TAU A G G R E G A T I O N I N T O A L Z H E I M E R P A I R E D HELICAL FILAMENTS
E X P R E S S I O N O F P A T H O G E N I C TAU, P301L, L E A D S T O M I C R O T U B U L E D I S R U P T I O N IN HIPPOCAMPAL NEURONS
Marcus Pickhardt*, Zuzanna Gazova, Martin von Bergen, Yipeng Wang, Inna Khlistounova, Antje Hascher, Jacek Biernat, Eva M. Mandelkow, Eckhard Mandelkow. Max-Planck-lnstitute, Hamburg, Germany. Contact
Linda M. Callahan*, Kenessa Edwards, Kathy A. Maguire-Zeiss, William J. Bowers, Wade C. Narrow, Susan Kang, Michael A. Mastrangelo, Rita E. Giuliano, Howard J. Federoff. University of Rochester, Rochester, NY,
e-mail: [email protected]
USA. Contact e-mail: [email protected]
Intracellular paired helical filaments (PHFs) comprising pathologically aggregated tau protein is one of the hallmarks of Alzheimer's disease and related tauopathies. It is thus expected that inhibition of tan polymerisation could help to prevent neuronal loss in these diseases. We have therefore searched for methods that allow testing of compounds that prevent the aggregation of tau, or that are able to induce the disassembly of aggregated tau. The assay is based on the fluorescence of the dye thioflavin S which changes when PHFs are formed. The assay has now been adapted to an automated high throughput format using 384-well plates. In a primary screen of a large compound library we have identified over 1200 compounds which are capable to inhibit PHF aggregation to an extent higher than 90%. From these 1200 compounds 77 were also able to disassemble PHFs with IC50 values in the IxM range. These results were confirmed by electron microscopy and by a recently established tryptophan fluorescence assay which enables us to monitor the aggregation state of the protein without adding an exogenous dye. The results show that PHF aggregation from recombinant tau protein is reversible and can be inhibited by low M W compounds. In microtubule binding experiments the compounds did not interfere with the stabilization of microtubules by tau. The future aim is to test these compounds in cell and animal models. For example, inducible expression of tan in a neuroblastoma cell line leads to incipient tan aggregation, as judged by the thioflavin S fluorescence assay and by Western blotting. This transformation can be prevented and even reversed by adding certain inhibitory compounds. Supported by Deutsche Forschungsgemeinschaft.
Background: The pathogenesis of abnormal tan in Alzheimer's disease is thought to include disruption of neuronal microtubules. Micrntubular disruption is predicted to lead to cellular dysfunction, including axonal transport dysfunction and synaptic loss. OBJECTIVE: We employ HSV amplicon technology to develop a model system in which the effects of pathogenic tan can be studied. Here we test the hypothesis that expression of pathogenic tau, P301L, produces a sequential disruption of microtubular networks in postmitotic neurons. Methods: El8 rat hippocampal neurons in co-culture were transduced at 14 DIV with HSV amplicons coexpressing either P301L tan, or normal human four-repeat tan with, the reporter gene, green fluorescent protein (GFP). A control HSV amplicon expressing GFP alone was utilized as a control. Three days foUowing transduction, cells were processed immunocytochemically with anti-acetylated tubulin to identify mature microtubules. Acetylated tubulin staining patterns were analyzed in a blinded quantitative analysis on 60X laser confocal images of GFP+ neurons imported into an MCID image analyzer. Morphometric measurements were performed using a camera-lucida like analysis. Results: We demonstrate that expression of P301L tan in postmitotic, mature neurons results in a profound disruption of the microtubule network within neuronal processes. The microtubule disruptions are in the form of small breaks and beads, most pronounced in primary and secondary processes. The disruption is not observed in mature neurons overexpressing normal four-repeat tan, nor is it seen in neurons expressing only GFP. Only mild alterations of mature microtubules are visible within somal microtubules despite strong P301L expression within the soma. Conclusions: We conclude the mechanism of pathogenic tan is mediated at least in part by signifcant disruption of the microtubular network created by numerous small breaks and beads within process microtubules. Our data indicate the effect of pathogenic tau expression differs for process microtubules as compared to somal microtubules. Our aim is to understand the sequence of upstream events leading to microtubule disruption within processes in neurons expressing abnormal tan, and in particular, the effects of pathogenic tau expression within neurons already undergoing aberrant amyloid processing.
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M O L E C U L A R M O T O R S I M P L I C A T E D IN T H E FAST A X O N A L T R A N S P O R T O F TAU
Wendy J. Noble*, MicheUe A. Litton, Brian H. Anderton, Diane P. Hanger.
Institute of Psychiatry, London, United Kingdom. Contact e-mail: [email protected]
Background: The axonally distributed microtubule-associated protein tan helps maintain cellular morphology and intracelhilar trafficking by stablising and promoting polymerisation of microtubules. Aberrant phosphorylation of tau leads to its aggregation and the accumulation of tan as inttacellular neurofibfillary tangles (NFTs), one of the pathological hallmarks of Alzheimer's disease (AD). Objective(s): We have investigated the mechanism of axonal transport of tan since misregulation of axonal transport has been implicated in the pathogenesis of AD and related disorders. Methods: Transport of EGFP-tagged tan in transfected rat cortical cultures was visualised in fxed cells and in live-cell video images. Immunofluorescence, co-immunoprecipitation and GST-binding were used to assess potential interactions of tan with motor proteins. Results: We found that transport of tan is dependent on an intact microtubule, but not actin, cytoskeleton in rat cortical neurons. Furthermore, as reported previously for neurnfilaments, tan moves at an overall slow rate of transport. However, live imaging of neurons reveals particulate, tau-containing slructures that can travel rapidly and bi-directionaUy, hut also spend long periods of time in a paused state. In transfected neurons, tan particles co-localise with the fast anterograde transport motor, kinesin, but not with the fast retrograde motor, dynein. Co-immunoprecipitation experiments demonstrate that tan may be part of a complex containing kinesin, but not dynein. In addition, we found a direct interaction of tan with GST-fusion proteins expressing kinesin light chains 1 and 2. Conclusions: Our results show that transport of tan is mediated, at least in part, via a direct interaction between tan and the fast anterograde motor kinesin in rat neurons. These findings have implications for AD, as well as other disorders in which tan deposition occurs, since defective axonal transport may be responsible for the accumulation of tau as fibfillar inclusions in affected neurons.
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N O N - H Y P E R P H O S P H O R Y L A T E D TAU A C C U M U L A T E S IN T H E P A T H O L O G I C A L H A L L M A R K S IN P R E S E N I L I N - 1 A L Z H E I M E R ' S DISEASE AND FRONTOTEMPORAL DEMENTIA
Claire E. Shepherd* 1,2, Emma C. Schofield 1.2, CJillian C. Gregory 1,2, Glenda M. Halliday 1,2 1Prince of Wales Medical Research Institute,
Sydney, Australia; 2 UNSW,, Sydney, Australia. Contact e-mail: c.shephe rd@un,wv,edu.au
Background: Changes in the phosphorylation state of the microtubule associated protein tau are thought to play a crucial role in the pathogenesis of a number of neurodegenerative disorders. Hyperphosphorylated tau in the form of neurofibrillary tangles and Pick bodies accumulate in the neuronal cell bodies in sporadic and presenilin-1 (PS-1) Alzheimer's disease (AD) and frontotemporal dementia (FIT)) respectively. Objective(s): To investigate the role of non-phosphorylated tan protein in AD and FTD. Methods: Cortical tissue was obtained from 8 sporadic AD, 6 presenilin-1 (PS-1) AD, 5 frontotemporal dementia (FI'D) and 10 control cases. Antibodies were used to detect total tan (tan-2) and hyperphosphorylated tau (AT8). Results: All of our PS-1 cases with mutations in and around exon 9 of the PS-1 gene deposited non-fibrillar, non-hyperphosphorylated tau within cotton wool plaques. In contrast, non-hyperphosphorylated tan was not seen in any of our sporadic AD cases or in our PS-1 cases with mutations in exons 5 and 6. In addition, all of our FTD cases deposited non-hyperphosphorylated tau in the microglia within the white matter but did not display any
~5-7]
Poster Session P3: Molecular Mechanisms of Neurodegeneration - Neurofibrillary Degeneration/Tau
SEARCH F O R S M A L L M O L E C U L E INHIBITORS OF
•
TAU A G G R E G A T I O N I N T O A L Z H E I M E R P A I R E D HELICAL FILAMENTS
E X P R E S S I O N O F P A T H O G E N I C TAU, P301L, L E A D S T O M I C R O T U B U L E D I S R U P T I O N IN HIPPOCAMPAL NEURONS
Marcus Pickhardt*, Zuzanna Gazova, Martin von Bergen, Yipeng Wang, Inna Khlistounova, Antje Hascher, Jacek Biernat, Eva M. Mandelkow, Eckhard Mandelkow. Max-Planck-lnstitute, Hamburg, Germany. Contact
Linda M. Callahan*, Kenessa Edwards, Kathy A. Maguire-Zeiss, William J. Bowers, Wade C. Narrow, Susan Kang, Michael A. Mastrangelo, Rita E. Giuliano, Howard J. Federoff. University of Rochester, Rochester, NY,
e-mail: [email protected]
USA. Contact e-mail: [email protected]
Intracellular paired helical filaments (PHFs) comprising pathologically aggregated tau protein is one of the hallmarks of Alzheimer's disease and related tauopathies. It is thus expected that inhibition of tan polymerisation could help to prevent neuronal loss in these diseases. We have therefore searched for methods that allow testing of compounds that prevent the aggregation of tau, or that are able to induce the disassembly of aggregated tau. The assay is based on the fluorescence of the dye thioflavin S which changes when PHFs are formed. The assay has now been adapted to an automated high throughput format using 384-well plates. In a primary screen of a large compound library we have identified over 1200 compounds which are capable to inhibit PHF aggregation to an extent higher than 90%. From these 1200 compounds 77 were also able to disassemble PHFs with IC50 values in the IxM range. These results were confirmed by electron microscopy and by a recently established tryptophan fluorescence assay which enables us to monitor the aggregation state of the protein without adding an exogenous dye. The results show that PHF aggregation from recombinant tau protein is reversible and can be inhibited by low M W compounds. In microtubule binding experiments the compounds did not interfere with the stabilization of microtubules by tau. The future aim is to test these compounds in cell and animal models. For example, inducible expression of tan in a neuroblastoma cell line leads to incipient tan aggregation, as judged by the thioflavin S fluorescence assay and by Western blotting. This transformation can be prevented and even reversed by adding certain inhibitory compounds. Supported by Deutsche Forschungsgemeinschaft.
Background: The pathogenesis of abnormal tan in Alzheimer's disease is thought to include disruption of neuronal microtubules. Micrntubular disruption is predicted to lead to cellular dysfunction, including axonal transport dysfunction and synaptic loss. OBJECTIVE: We employ HSV amplicon technology to develop a model system in which the effects of pathogenic tan can be studied. Here we test the hypothesis that expression of pathogenic tau, P301L, produces a sequential disruption of microtubular networks in postmitotic neurons. Methods: El8 rat hippocampal neurons in co-culture were transduced at 14 DIV with HSV amplicons coexpressing either P301L tan, or normal human four-repeat tan with, the reporter gene, green fluorescent protein (GFP). A control HSV amplicon expressing GFP alone was utilized as a control. Three days foUowing transduction, cells were processed immunocytochemically with anti-acetylated tubulin to identify mature microtubules. Acetylated tubulin staining patterns were analyzed in a blinded quantitative analysis on 60X laser confocal images of GFP+ neurons imported into an MCID image analyzer. Morphometric measurements were performed using a camera-lucida like analysis. Results: We demonstrate that expression of P301L tan in postmitotic, mature neurons results in a profound disruption of the microtubule network within neuronal processes. The microtubule disruptions are in the form of small breaks and beads, most pronounced in primary and secondary processes. The disruption is not observed in mature neurons overexpressing normal four-repeat tan, nor is it seen in neurons expressing only GFP. Only mild alterations of mature microtubules are visible within somal microtubules despite strong P301L expression within the soma. Conclusions: We conclude the mechanism of pathogenic tan is mediated at least in part by signifcant disruption of the microtubular network created by numerous small breaks and beads within process microtubules. Our data indicate the effect of pathogenic tau expression differs for process microtubules as compared to somal microtubules. Our aim is to understand the sequence of upstream events leading to microtubule disruption within processes in neurons expressing abnormal tan, and in particular, the effects of pathogenic tau expression within neurons already undergoing aberrant amyloid processing.
•
M O L E C U L A R M O T O R S I M P L I C A T E D IN T H E FAST A X O N A L T R A N S P O R T O F TAU
Wendy J. Noble*, MicheUe A. Litton, Brian H. Anderton, Diane P. Hanger.
Institute of Psychiatry, London, United Kingdom. Contact e-mail: [email protected]
Background: The axonally distributed microtubule-associated protein tan helps maintain cellular morphology and intracelhilar trafficking by stablising and promoting polymerisation of microtubules. Aberrant phosphorylation of tau leads to its aggregation and the accumulation of tan as inttacellular neurofibfillary tangles (NFTs), one of the pathological hallmarks of Alzheimer's disease (AD). Objective(s): We have investigated the mechanism of axonal transport of tan since misregulation of axonal transport has been implicated in the pathogenesis of AD and related disorders. Methods: Transport of EGFP-tagged tan in transfected rat cortical cultures was visualised in fxed cells and in live-cell video images. Immunofluorescence, co-immunoprecipitation and GST-binding were used to assess potential interactions of tan with motor proteins. Results: We found that transport of tan is dependent on an intact microtubule, but not actin, cytoskeleton in rat cortical neurons. Furthermore, as reported previously for neurnfilaments, tan moves at an overall slow rate of transport. However, live imaging of neurons reveals particulate, tau-containing slructures that can travel rapidly and bi-directionaUy, hut also spend long periods of time in a paused state. In transfected neurons, tan particles co-localise with the fast anterograde transport motor, kinesin, but not with the fast retrograde motor, dynein. Co-immunoprecipitation experiments demonstrate that tan may be part of a complex containing kinesin, but not dynein. In addition, we found a direct interaction of tan with GST-fusion proteins expressing kinesin light chains 1 and 2. Conclusions: Our results show that transport of tan is mediated, at least in part, via a direct interaction between tan and the fast anterograde motor kinesin in rat neurons. These findings have implications for AD, as well as other disorders in which tan deposition occurs, since defective axonal transport may be responsible for the accumulation of tau as fibfillar inclusions in affected neurons.
•
N O N - H Y P E R P H O S P H O R Y L A T E D TAU A C C U M U L A T E S IN T H E P A T H O L O G I C A L H A L L M A R K S IN P R E S E N I L I N - 1 A L Z H E I M E R ' S DISEASE AND FRONTOTEMPORAL DEMENTIA
Claire E. Shepherd* 1,2, Emma C. Schofield 1.2, CJillian C. Gregory 1,2, Glenda M. Halliday 1,2 1Prince of Wales Medical Research Institute,
Sydney, Australia; 2 UNSW,, Sydney, Australia. Contact e-mail: c.shephe rd@un,wv,edu.au
Background: Changes in the phosphorylation state of the microtubule associated protein tau are thought to play a crucial role in the pathogenesis of a number of neurodegenerative disorders. Hyperphosphorylated tau in the form of neurofibrillary tangles and Pick bodies accumulate in the neuronal cell bodies in sporadic and presenilin-1 (PS-1) Alzheimer's disease (AD) and frontotemporal dementia (FIT)) respectively. Objective(s): To investigate the role of non-phosphorylated tan protein in AD and FTD. Methods: Cortical tissue was obtained from 8 sporadic AD, 6 presenilin-1 (PS-1) AD, 5 frontotemporal dementia (FI'D) and 10 control cases. Antibodies were used to detect total tan (tan-2) and hyperphosphorylated tau (AT8). Results: All of our PS-1 cases with mutations in and around exon 9 of the PS-1 gene deposited non-fibrillar, non-hyperphosphorylated tau within cotton wool plaques. In contrast, non-hyperphosphorylated tan was not seen in any of our sporadic AD cases or in our PS-1 cases with mutations in exons 5 and 6. In addition, all of our FTD cases deposited non-hyperphosphorylated tau in the microglia within the white matter but did not display any