- Email: [email protected]
O4-01-03: Dissecting tau-mediated toxicity in novel Tau transgenic mouse and tissue culture models
T184
Oral O4-01: Animal and Cellular Models 2
pathologic processes we generated inducible transgenic mouse lines, which express either the 4-repeat tau domain with the FTDP-17 mutation Delta-K280 (TauRD/DeltaK280 - “pro-aggregation mutant”) or the 4-repeat tau domain with Delta-K280 deletion and two proline mutations in the hexapeptide motifs (TauRD/DeltaK280/I277P/I308 - “antiaggregation mutant”). Results: The DeltaK280 mutation accelerates the aggregation of tau, but the inserted proline residues inhibit the tau aggregation in vitro and in cell models. Inducible transgene expression in mice was driven by a forebrain-specific CaMKII promoter in a Tet-Off system and can be suppressed by doxycycline. The pro-aggregation mutant showed aggregated tau in sarkosyl insoluble fractions and Gallyas silver stained neurofibrillary tangles from 3 months onwards, even though the level of the human tau protein was lower than endogenous mouse tau. Tau preparations from pro-aggregation mutant mice revealed PHFs by electron microscopy. Consistent with the tau pathology the neuronal loss was age-dependent and visible in the dentate gyrus as early as 5 months. The immunohistochemisty results showed phosphorylated tau at S262 missorted into the somatodendritic compartment of cortical and hippocampal neurons. The anti-aggregation mutant with a similar expression level as the pro-aggregation mutant did not show aggregated tau or neuronal loss, but missorting into the somatodendritic compartment. The level of synaptophysin, a presynaptic marker and the number of spine-synapses were reduced in the stratum radiatum of the pro-aggregation mutant, but not of the antiaggregation mutant. Six weeks of switching off the tau transgene in the pro-aggregation mutant lead to ⬃90% reduction in the level of soluble human tau protein, to complete reversal of the pathological somatodendritic localization and of phosphorylation in the repeat domain. Remarkably, the aggregation was only partly reversed. The remaining aggregates did not contain the exogenous human tau, but the endogenous mouse tau. Conclusions: The results argue that a toxic species of tau, once introduced into neurons, can “poison” endogenous tau and propagate its aggregation for extended time periods. Supported by MPG and DFG. O4-01-03
DISSECTING TAU-MEDIATED TOXICITY IN NOVEL TAU TRANSGENIC MOUSE AND TISSUE CULTURE MODELS
Jurgen Gotz1, Nicole Schonrock1, Andreas Wiesner1, Yazi Ke1, Laita Bokhari1, Yun-An Lim1, Natasha Deters1, Anne Eckert2, Christian Czech3, Steven Pelech4, Lars M. Ittner1, 1Brain and Mind Research Institute, University of Sydney, Camperdown, Australia; 2 Neurobiology Research Laboratory, Psychiatric University Clinic Basel, Basel, Switzerland; 3F. Hoffmann-La Roche AG, CNS Research, Basel, Switzerland; 4Kinexus Bioinformatics Corporation, Vancouver, BC, Canada. Contact e-mail: [email protected] Background: The role of tau in neurodegeneration is far from understood, however, we have shown previously using transgenic mice that Abeta can augment a tau pathology, that tau and Abeta act synergistically in activating cell cycle-regulated genes and that tau aggregation causes mitochondrial dysfunction. Applying proteomics to Abeta-injected P301L mice and Abeta-treated tau-expressing human neuroblastoma cells identified deregulated proteins in the categories stress response and cytoskeletal organization. We could validate our findings both functionally and on human AD tissue. Methods: Our initial proteomics screen in P301L mice had indicated an upregulation of synaptic proteins. Extending these studies, we enriched for neurosynaptosomes of four strains, P301L, Abeta-producing APP-PS2, triple transgenic and wild-type mice. We obtained tryptic digests and labelled them separately with isobaric iTRAQ tags, allowing for a comparative quantification. Furthermore, to dissect the role of tau in neurodegeneration we established novel transgenic animal models. Results: In the iTRAQ screen, we identified a total of more than 1200 proteins of which 111 were altered in the presence of plaques or tangles or both. They are currently validated. Of the new transgenic strains, one expresses K369I
mutant tau and models memory impairment and Parkinsonism. We were able to dissect the underlying pathomechanism at a molecular level and found a pathological protein interaction causing the phenotype. Another novel mouse strain expresses only the projection domain of tau (deltaTau). We found that deltaTau not only localized to the soma, but entered the axon and was transported to the growth cone, in the absence of a microtubule-binding domain. Transport did not require the presence of endogenous murine tau. This indicates that axonal transport of tau is not solely mediated by binding to microtubules. DeltaTau is localized to the membrane and unlike full-length wild-type and mutant tau, is only weakly phosphorylated. The deltaTau mice showed a mitochondrial phenotype similar to the P301L and K369I mice. We since crossed the deltaTau mice with P301L mice to determine whether the NFT phenotype in P301L mice is enhanced. Conclusions: These proteomics screens together with the generation of novel mouse strains have proved insight into pathomechanisms of neurodegenerative diseases. O4-01-04
MITOCHONDRIAL CLUMPING IS A CENTRAL FEATURE IN THE TAU NEURODEGENERATION MECHANISM IN AN IN SITU TAUOPATHY MODEL
Garth F. Hall1, Sangmook Lee1, Adriana Ferreira2, Gloria Lee3, 1U. Mass. Lowell, Lowell, MA, USA; 2Northwestern University, Chicago, IL, USA; 3University of Iowa, Iowa City, IA, USA. Contact e-mail: [email protected] Background: Exonic point mutations in the human tau (htau) coding sequence that cause tauopathy in humans have been shown to induce or accelerate neurodegeneration in a variety of transgenic animal models. Both wild type and mutant htau isoforms also induce neurodegeneration in identified giant neurons (ABCs) in the brainstem of the ammocoete sea lamprey (Petromyzon marinus) that have been microinjected with plasmids encoding htau isoforms. While recent studies suggest that htau modifications ( hyperphosphorylation, aggregation or truncation), or abnormal interactions between htau and microtubule motor proteins may be important in the cellular mechanisms that result in tauopathy, little is known about the precise roles and spatiotemporal sequence of these changes and events in the mechanism by which htau induces neurodegeneration, especially in neurons in situ. Methods: We have used confocal microscopic analysis of double-immunolabeled transverse sections through ABC dendrites in this study. Results: We have previously shown that chronic htau expression induces progressive centripetal degeneration in ABC dendrites, with the distalmost dendrites first becoming swollen and beaded and then degenerating into isolated fragments and disappearing entirely. This sequence of events is accompanied by the abnormal aggregation of membrane bound organelles, microtubule and synaptic loss, and the formation of htau filaments and autophagosome-like structures in the affected dendrites. Dendritic degeneration always moves proximally along ABC dendrites with time, and thus provides a sequential record of events that offers clues to the mechanism of htau-induced neurodegeneraton in situ. Bead formation is spatiotemporally correlated with both focal microtubule destabilization and the clumping of mitochondria in degenerating ABC dendrites. Mitochondrial clumping occurs proximal to the point at which dendritic beading begins, suggesting that it precedes dendritic beading and may play a role in causing it. Moreover, expression of the highly toxic calpain cleavage product of htau40 (residues 45-230) strongly exacerbates mitochondrial clumping throughout ABC dendrites while causing the simultaneous initiation of beading in proximal as well as distal dendrites. Conclusions: We propose that mitochondrial maldistribution plays an important role in mechanism of the neurodegenerative events induced by htau expression in ABCs, and that it may also be important in the cytopathogenesis of human tauopathies.
Oral O4-01: Animal and Cellular Models 2
pathologic processes we generated inducible transgenic mouse lines, which express either the 4-repeat tau domain with the FTDP-17 mutation Delta-K280 (TauRD/DeltaK280 - “pro-aggregation mutant”) or the 4-repeat tau domain with Delta-K280 deletion and two proline mutations in the hexapeptide motifs (TauRD/DeltaK280/I277P/I308 - “antiaggregation mutant”). Results: The DeltaK280 mutation accelerates the aggregation of tau, but the inserted proline residues inhibit the tau aggregation in vitro and in cell models. Inducible transgene expression in mice was driven by a forebrain-specific CaMKII promoter in a Tet-Off system and can be suppressed by doxycycline. The pro-aggregation mutant showed aggregated tau in sarkosyl insoluble fractions and Gallyas silver stained neurofibrillary tangles from 3 months onwards, even though the level of the human tau protein was lower than endogenous mouse tau. Tau preparations from pro-aggregation mutant mice revealed PHFs by electron microscopy. Consistent with the tau pathology the neuronal loss was age-dependent and visible in the dentate gyrus as early as 5 months. The immunohistochemisty results showed phosphorylated tau at S262 missorted into the somatodendritic compartment of cortical and hippocampal neurons. The anti-aggregation mutant with a similar expression level as the pro-aggregation mutant did not show aggregated tau or neuronal loss, but missorting into the somatodendritic compartment. The level of synaptophysin, a presynaptic marker and the number of spine-synapses were reduced in the stratum radiatum of the pro-aggregation mutant, but not of the antiaggregation mutant. Six weeks of switching off the tau transgene in the pro-aggregation mutant lead to ⬃90% reduction in the level of soluble human tau protein, to complete reversal of the pathological somatodendritic localization and of phosphorylation in the repeat domain. Remarkably, the aggregation was only partly reversed. The remaining aggregates did not contain the exogenous human tau, but the endogenous mouse tau. Conclusions: The results argue that a toxic species of tau, once introduced into neurons, can “poison” endogenous tau and propagate its aggregation for extended time periods. Supported by MPG and DFG. O4-01-03
DISSECTING TAU-MEDIATED TOXICITY IN NOVEL TAU TRANSGENIC MOUSE AND TISSUE CULTURE MODELS
Jurgen Gotz1, Nicole Schonrock1, Andreas Wiesner1, Yazi Ke1, Laita Bokhari1, Yun-An Lim1, Natasha Deters1, Anne Eckert2, Christian Czech3, Steven Pelech4, Lars M. Ittner1, 1Brain and Mind Research Institute, University of Sydney, Camperdown, Australia; 2 Neurobiology Research Laboratory, Psychiatric University Clinic Basel, Basel, Switzerland; 3F. Hoffmann-La Roche AG, CNS Research, Basel, Switzerland; 4Kinexus Bioinformatics Corporation, Vancouver, BC, Canada. Contact e-mail: [email protected] Background: The role of tau in neurodegeneration is far from understood, however, we have shown previously using transgenic mice that Abeta can augment a tau pathology, that tau and Abeta act synergistically in activating cell cycle-regulated genes and that tau aggregation causes mitochondrial dysfunction. Applying proteomics to Abeta-injected P301L mice and Abeta-treated tau-expressing human neuroblastoma cells identified deregulated proteins in the categories stress response and cytoskeletal organization. We could validate our findings both functionally and on human AD tissue. Methods: Our initial proteomics screen in P301L mice had indicated an upregulation of synaptic proteins. Extending these studies, we enriched for neurosynaptosomes of four strains, P301L, Abeta-producing APP-PS2, triple transgenic and wild-type mice. We obtained tryptic digests and labelled them separately with isobaric iTRAQ tags, allowing for a comparative quantification. Furthermore, to dissect the role of tau in neurodegeneration we established novel transgenic animal models. Results: In the iTRAQ screen, we identified a total of more than 1200 proteins of which 111 were altered in the presence of plaques or tangles or both. They are currently validated. Of the new transgenic strains, one expresses K369I
mutant tau and models memory impairment and Parkinsonism. We were able to dissect the underlying pathomechanism at a molecular level and found a pathological protein interaction causing the phenotype. Another novel mouse strain expresses only the projection domain of tau (deltaTau). We found that deltaTau not only localized to the soma, but entered the axon and was transported to the growth cone, in the absence of a microtubule-binding domain. Transport did not require the presence of endogenous murine tau. This indicates that axonal transport of tau is not solely mediated by binding to microtubules. DeltaTau is localized to the membrane and unlike full-length wild-type and mutant tau, is only weakly phosphorylated. The deltaTau mice showed a mitochondrial phenotype similar to the P301L and K369I mice. We since crossed the deltaTau mice with P301L mice to determine whether the NFT phenotype in P301L mice is enhanced. Conclusions: These proteomics screens together with the generation of novel mouse strains have proved insight into pathomechanisms of neurodegenerative diseases. O4-01-04
MITOCHONDRIAL CLUMPING IS A CENTRAL FEATURE IN THE TAU NEURODEGENERATION MECHANISM IN AN IN SITU TAUOPATHY MODEL
Garth F. Hall1, Sangmook Lee1, Adriana Ferreira2, Gloria Lee3, 1U. Mass. Lowell, Lowell, MA, USA; 2Northwestern University, Chicago, IL, USA; 3University of Iowa, Iowa City, IA, USA. Contact e-mail: [email protected] Background: Exonic point mutations in the human tau (htau) coding sequence that cause tauopathy in humans have been shown to induce or accelerate neurodegeneration in a variety of transgenic animal models. Both wild type and mutant htau isoforms also induce neurodegeneration in identified giant neurons (ABCs) in the brainstem of the ammocoete sea lamprey (Petromyzon marinus) that have been microinjected with plasmids encoding htau isoforms. While recent studies suggest that htau modifications ( hyperphosphorylation, aggregation or truncation), or abnormal interactions between htau and microtubule motor proteins may be important in the cellular mechanisms that result in tauopathy, little is known about the precise roles and spatiotemporal sequence of these changes and events in the mechanism by which htau induces neurodegeneration, especially in neurons in situ. Methods: We have used confocal microscopic analysis of double-immunolabeled transverse sections through ABC dendrites in this study. Results: We have previously shown that chronic htau expression induces progressive centripetal degeneration in ABC dendrites, with the distalmost dendrites first becoming swollen and beaded and then degenerating into isolated fragments and disappearing entirely. This sequence of events is accompanied by the abnormal aggregation of membrane bound organelles, microtubule and synaptic loss, and the formation of htau filaments and autophagosome-like structures in the affected dendrites. Dendritic degeneration always moves proximally along ABC dendrites with time, and thus provides a sequential record of events that offers clues to the mechanism of htau-induced neurodegeneraton in situ. Bead formation is spatiotemporally correlated with both focal microtubule destabilization and the clumping of mitochondria in degenerating ABC dendrites. Mitochondrial clumping occurs proximal to the point at which dendritic beading begins, suggesting that it precedes dendritic beading and may play a role in causing it. Moreover, expression of the highly toxic calpain cleavage product of htau40 (residues 45-230) strongly exacerbates mitochondrial clumping throughout ABC dendrites while causing the simultaneous initiation of beading in proximal as well as distal dendrites. Conclusions: We propose that mitochondrial maldistribution plays an important role in mechanism of the neurodegenerative events induced by htau expression in ABCs, and that it may also be important in the cytopathogenesis of human tauopathies.