- Email: [email protected]
Viral delivery of mutant tau drives tauopathy in vivo
P138
Podium Presentations: Sunday, July 19, 2015
SUNDAY, JULY 19, 2015 ORAL SESSIONS O1-06 DEVELOPMENT OF NEW MODELS: MODELLING TAUOPATHY IN VITRO, IN VIVO, AND IN MAN O1-06-01
MODELING TAUOPATHIES IN HUMAN PLURIPOTENT STEM CELLS
Celeste Karch1, Rita Martinez1, Simon Hsu1, Lubov Ezerskiy1, Joanne Norton1, Alison Goate2,3, 1Washington University in St. Louis, St. Louis, MO, USA; 2Mt. Sinai School of Medicine, New York, NY, USA; 3 Knight Alzheimer Disease Research Center, Saint Louis, MO, USA. Contact e-mail: [email protected] Background: Tauopathies are a class of neurodegenerative diseases
that are characterized by hyperphosphorylated tau aggregates in the brain. In a subset of tauopathies, genetic changes in MAPT, the gene encoding the tau protein, are sufficient to initiate a cascade of events that leads to tau aggregation and death of neuronal populations in the brain. Increasing evidence suggests that tau aggregates spread along neuronal networks in the brain via tau release and tau uptake. However, the cascade of events that leads to disease remains poorly understood. Methods: We sought to develop a stem cell model of tauopathies that captures the genetic complexities of the MAPT gene and the phenotypic complexities of the human neuron. We generated human induced pluripotent stem cell (iPSC) using nonintegrating Sendai virus from dermal fibroblasts carrying MAPT mutations (P301L, IVS10+16, V337M, R406W). We measured tau phosphorylation, release and aggregation in iPSC-derived neurons from MAPT mutation carriers and controls. To determine if correction of MAPT mutations restores normal tau metabolism, we developed a pipeline for efficient, seamless modification of point mutations using CRISPR/Cas9 technology. Applying this pipeline to a MAPT P301L iPSC line, we generated isogenic, iPSC clones that are corrected to WT or that are modified to MAPTP301S. Results: Consistent with our findings in immortalized cell models, we found that tau is actively released from human iPSC-derived neurons via the unconventional secretory pathway and that calcium signaling plays an important role in tau release. We found that the rate of tau release was modified in iPSC-derived neurons from MAPT mutation carriers compared with controls. Reversion of the MAPT P301L to WT using CRISPR/Cas9 restored the tau release rate to the rates observed in unrelated control lines. Additionally, human iPSCderived neurons endogenously expressing MAPTmutations produced multimeric tau species that were absent in unrelated and isogenic control lines. Conclusions: Together, these stem cell models capture key pathological hallmarks of Alzheimer’s disease and other tauopathies and, thus, are beginning to define the cascade of events that lead to neurodegenerative tauopathies, providing avenues for advancement in therapeutic intervention. O1-06-02
VIRAL DELIVERY OF MUTANT TAU DRIVES TAUOPATHY IN VIVO
Leonard Petrucelli, Casey Cook, Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: Aberrant accumulation of the tau protein drives the formation of neurofibrillary tangles (NFTs), though efforts to elucidate pathogenic mechanisms and assess the efficacy of various therapeutic targets are limited by constraints of existing models of tauopathy. Methods: Somatic brain transgenesis was utilized to deliver adeno-associated virus (AAV) encoding human mutant P301L-tau to the brain of mice on postnatal day 0. Results: We
observe widespread expression of human tau at 6 months of age, along with the accumulation of hyperphosphorylated and abnormally-folded proteinase K-resistant tau species. Neurofibrillary pathology is also detected with Gallyas silver stain and Thioflavin S, and NFTs composed of closely-packed filaments are observed by electron microscopy. In addition to classic markers of pathological tau, significant neuroinflammatory changes and extensive gliosis are detected in AAV-TauP301L mice. This novel AAV-based model of tauopathy also recapitulates the behavioral phenotype characteristic of other mouse models of tauopathy, including abnormalities in exploration, anxiety, sociability, and learning and memory. Conclusions: These data indicate that biochemical and neuropathological hallmarks of tauopathies are accurately conserved in this novel AAV-based model of tauopathy, a model which offers exceptional versatility in comparison to existing transgenic models. Therefore we anticipate this approach will facilitate the identification and validation of genetic modifiers of disease, as well as accelerate preclinical assessment of potential therapeutic targets, such as HDAC6. O1-06-03
ACUTE MODELS OF TAU HYPERPHOSPHORYLATION
Cornelia Schweinzer, Nicole Taub, Daniel Havas, Ewald Auer, Martina Farcher, Birgit Hutter-Paier, QPS Austria, Grambach, Austria. Contact e-mail: [email protected] Background: Since Tau hyperphosphorylation in the central nervous system (CNS) plays a major role in many neurodegenerative diseases as Alzheimer’s disease and tauopathies, acute cellular and in vivo models represent inevitable tools to screen potential promising drug candidates. Since hypothermia has been discussed as to trigger Tau hyperphosphorylation, we aimed to investigate Tau phosphorylation profiles parallel in cellular and murine systems under hypothermic conditions. Methods: Hypothermia was either induced via pentobarbital injection in wildtype mice or hypothermic incubation of SH-SY5Y or SH-SY5Y overexpressing the longest isoform of human Tau 441 carrying two well-characterized mutations (SH-Tau) cells. Subsequently, total Tau and its phosphorylated species were analyzed in brains and cellular lysates. Moreover, evaluations of pGSK3 beta and beta amyloid levels were performed. Results: In vivo, hypothermia triggered hyperphosphorylation of various Tau sites such as Ser396, Ser262 and Thr181 most likely via enhanced GSK3beta activity. Total Tau levels remained unaltered, whereas amyloid beta levels were significantly increased. In vitro, hypothermia-induced effects on Tau hyperphosphorylation were more prominent in SH-Tau than in SH cells pointing towards the need of human mutated Tau to enlarge the therapeutic window. As in mice, total Tau levels stayed unaltered, whereas pSer396 and pSer262 Tau levels were significantly enhanced in SH and even more pronounced in SH-Tau cells under hypothermic conditions. LiCl, a known kinase inhibitor reversed the hypothermia-induced hyperphosphorylation. Conclusions: Hypothermia in wildtype mice and SH-Tau cells triggers hyperphosphorylation and mimics pathogenic events found in various neurodegenerative diseases. Thus both models are useful and acute models to study CNS drug mechanisms. O1-06-04 1
NON-HUMAN PRIMATE MODEL OF TAUOPATHY
Luc Buee , Morvane Colin1, Simon Dujardin1, Romina Aron Badin2, Alexis-Pierre Bemelmans2, Caroline Jan2, Nadja Van Camp2, Nicole Deglon3, Philippe Hantraye2, 1Inserm, University of Lille, Lille, France; 2CEA CNRS URA2210, Fontenay-aux-Roses, France; 3Laboratoire
Podium Presentations: Sunday, July 19, 2015
SUNDAY, JULY 19, 2015 ORAL SESSIONS O1-06 DEVELOPMENT OF NEW MODELS: MODELLING TAUOPATHY IN VITRO, IN VIVO, AND IN MAN O1-06-01
MODELING TAUOPATHIES IN HUMAN PLURIPOTENT STEM CELLS
Celeste Karch1, Rita Martinez1, Simon Hsu1, Lubov Ezerskiy1, Joanne Norton1, Alison Goate2,3, 1Washington University in St. Louis, St. Louis, MO, USA; 2Mt. Sinai School of Medicine, New York, NY, USA; 3 Knight Alzheimer Disease Research Center, Saint Louis, MO, USA. Contact e-mail: [email protected] Background: Tauopathies are a class of neurodegenerative diseases
that are characterized by hyperphosphorylated tau aggregates in the brain. In a subset of tauopathies, genetic changes in MAPT, the gene encoding the tau protein, are sufficient to initiate a cascade of events that leads to tau aggregation and death of neuronal populations in the brain. Increasing evidence suggests that tau aggregates spread along neuronal networks in the brain via tau release and tau uptake. However, the cascade of events that leads to disease remains poorly understood. Methods: We sought to develop a stem cell model of tauopathies that captures the genetic complexities of the MAPT gene and the phenotypic complexities of the human neuron. We generated human induced pluripotent stem cell (iPSC) using nonintegrating Sendai virus from dermal fibroblasts carrying MAPT mutations (P301L, IVS10+16, V337M, R406W). We measured tau phosphorylation, release and aggregation in iPSC-derived neurons from MAPT mutation carriers and controls. To determine if correction of MAPT mutations restores normal tau metabolism, we developed a pipeline for efficient, seamless modification of point mutations using CRISPR/Cas9 technology. Applying this pipeline to a MAPT P301L iPSC line, we generated isogenic, iPSC clones that are corrected to WT or that are modified to MAPTP301S. Results: Consistent with our findings in immortalized cell models, we found that tau is actively released from human iPSC-derived neurons via the unconventional secretory pathway and that calcium signaling plays an important role in tau release. We found that the rate of tau release was modified in iPSC-derived neurons from MAPT mutation carriers compared with controls. Reversion of the MAPT P301L to WT using CRISPR/Cas9 restored the tau release rate to the rates observed in unrelated control lines. Additionally, human iPSCderived neurons endogenously expressing MAPTmutations produced multimeric tau species that were absent in unrelated and isogenic control lines. Conclusions: Together, these stem cell models capture key pathological hallmarks of Alzheimer’s disease and other tauopathies and, thus, are beginning to define the cascade of events that lead to neurodegenerative tauopathies, providing avenues for advancement in therapeutic intervention. O1-06-02
VIRAL DELIVERY OF MUTANT TAU DRIVES TAUOPATHY IN VIVO
Leonard Petrucelli, Casey Cook, Mayo Clinic, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: Aberrant accumulation of the tau protein drives the formation of neurofibrillary tangles (NFTs), though efforts to elucidate pathogenic mechanisms and assess the efficacy of various therapeutic targets are limited by constraints of existing models of tauopathy. Methods: Somatic brain transgenesis was utilized to deliver adeno-associated virus (AAV) encoding human mutant P301L-tau to the brain of mice on postnatal day 0. Results: We
observe widespread expression of human tau at 6 months of age, along with the accumulation of hyperphosphorylated and abnormally-folded proteinase K-resistant tau species. Neurofibrillary pathology is also detected with Gallyas silver stain and Thioflavin S, and NFTs composed of closely-packed filaments are observed by electron microscopy. In addition to classic markers of pathological tau, significant neuroinflammatory changes and extensive gliosis are detected in AAV-TauP301L mice. This novel AAV-based model of tauopathy also recapitulates the behavioral phenotype characteristic of other mouse models of tauopathy, including abnormalities in exploration, anxiety, sociability, and learning and memory. Conclusions: These data indicate that biochemical and neuropathological hallmarks of tauopathies are accurately conserved in this novel AAV-based model of tauopathy, a model which offers exceptional versatility in comparison to existing transgenic models. Therefore we anticipate this approach will facilitate the identification and validation of genetic modifiers of disease, as well as accelerate preclinical assessment of potential therapeutic targets, such as HDAC6. O1-06-03
ACUTE MODELS OF TAU HYPERPHOSPHORYLATION
Cornelia Schweinzer, Nicole Taub, Daniel Havas, Ewald Auer, Martina Farcher, Birgit Hutter-Paier, QPS Austria, Grambach, Austria. Contact e-mail: [email protected] Background: Since Tau hyperphosphorylation in the central nervous system (CNS) plays a major role in many neurodegenerative diseases as Alzheimer’s disease and tauopathies, acute cellular and in vivo models represent inevitable tools to screen potential promising drug candidates. Since hypothermia has been discussed as to trigger Tau hyperphosphorylation, we aimed to investigate Tau phosphorylation profiles parallel in cellular and murine systems under hypothermic conditions. Methods: Hypothermia was either induced via pentobarbital injection in wildtype mice or hypothermic incubation of SH-SY5Y or SH-SY5Y overexpressing the longest isoform of human Tau 441 carrying two well-characterized mutations (SH-Tau) cells. Subsequently, total Tau and its phosphorylated species were analyzed in brains and cellular lysates. Moreover, evaluations of pGSK3 beta and beta amyloid levels were performed. Results: In vivo, hypothermia triggered hyperphosphorylation of various Tau sites such as Ser396, Ser262 and Thr181 most likely via enhanced GSK3beta activity. Total Tau levels remained unaltered, whereas amyloid beta levels were significantly increased. In vitro, hypothermia-induced effects on Tau hyperphosphorylation were more prominent in SH-Tau than in SH cells pointing towards the need of human mutated Tau to enlarge the therapeutic window. As in mice, total Tau levels stayed unaltered, whereas pSer396 and pSer262 Tau levels were significantly enhanced in SH and even more pronounced in SH-Tau cells under hypothermic conditions. LiCl, a known kinase inhibitor reversed the hypothermia-induced hyperphosphorylation. Conclusions: Hypothermia in wildtype mice and SH-Tau cells triggers hyperphosphorylation and mimics pathogenic events found in various neurodegenerative diseases. Thus both models are useful and acute models to study CNS drug mechanisms. O1-06-04 1
NON-HUMAN PRIMATE MODEL OF TAUOPATHY
Luc Buee , Morvane Colin1, Simon Dujardin1, Romina Aron Badin2, Alexis-Pierre Bemelmans2, Caroline Jan2, Nadja Van Camp2, Nicole Deglon3, Philippe Hantraye2, 1Inserm, University of Lille, Lille, France; 2CEA CNRS URA2210, Fontenay-aux-Roses, France; 3Laboratoire